Tyrosine kinase receptor antagonists and methods of treatment for pancreatic cancer

ABSTRACT

A method of treatment is disclosed whereby cancer cells are brought into contact with a formulation comprising an inhibitor of tyrosine kinase receptors. The formulation may be comprised of an injectable carrier and two or more tyrosine kinase receptor inhibitors which may be nordihydrogluaiaretic acid (NDGA) and doxyrubicine.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional Application Nos.60/731,384 filed Oct. 28, 2005; 60/825,663 filed Sep. 14, 2006; and60/828,937 filed Oct. 10, 2006, which applications are incorporatedherein by reference.

BACKGROUND OF THE INVENTION

Many tumor cells depend on the activity of tyrosine kinases, which act,among other functions, to depress apoptosis in the cell. The tyrosinekinases are usually overproduced in malignant cells, which contributesto the cell's ability to resist apoptosis. Modulating the activity ofthese proteins provides an effective means of treating cancer while notunduly damaging normal tissues. For example, about 25% of breast tumorsexpress unusually high levels of the Her2 protein, a tyrosine kinasereceptor that normally plays a part in the development of the mammaryepithelium. Herceptin® (Trastuzumab) is a humanized antibody that iscurrently used to treat breast cancer by targeting and blocking thefunction of the Her2 protein. Other treatments focus on interfering withthe receptors to overexpressed tyrosine kinase proteins. Receptorsinclude HER2/neu and IGF-1R. See, Meric et al. (Apr. 2002) J. Am. Coll.Surg. 194(4):488-501.

The major lignin in chaparral, known as nordihydroguaiaretic acid (NDGA)is a potent antioxidant and was originally used in commercial foodproducts as a preservative. See, U.S. Pat. No. 2,644,822. Later, it wasdiscovered that NDGA is useful in the treatment of diabetes. Hsu et al.(2001) Cell Transplant. 10(3):255-262. More recently, NDGA wasinvestigated as a treatment for cancer because it inhibits the plateletderived growth factor receptor and the protein kinase C intracellularsignalling family, which both play an important role in proliferationand survival of cancers. Moreover, NDGA induces apoptosis in tumorxenografts. Although it is likely to have several targets of action,NDGA is well tolerated in animals. However, high concentrations of NDGAare required for efficacy and it has been suggested that more potentanalogs may be required. See, McDonald et al. (2001) Anticancer DrugDes. 16(6):261-270.

Other cancer drugs include doxorubicin hydrochloride (DOX), which isused alone or in combination with other drugs for treatment of malignantlymphomas and leukemias. DOX is believed to bind DNA and inhibit nucleicacid synthesis. Examples of tumors amenable to treatment with DOX areacute lymphoblastic leukemia, acute myeloblastic leukemia, Wilm's tumor,soft tissue and bone sarcomas, breast carcinoma and ovarian carcinoma.The dosage needs to be closely monitored because it can causeirreversible cardiac damage. A typical dose for adults, when givenintravenously is 60-75 mg/m2 once in 21 days, or 30 mg/m2 daily for 3days every four weeks, where the total cumulative dose should not exceed550 mg/m2 without monitoring for cardiac function.

It is well established that breast cancer is regulated by receptors forthe female sex steroids, estrogen and progesterone. It is nowappreciated that receptor tyrosine kinases (RTKs) are also veryimportant for breast cancer growth (Arteaga C L, Moulder S L, Yakes F M:HER (erbB) tyrosine kinase inhibitors in the treatment of breast cancer.Semin Oncol 29:4-10, 2002; Averbuch S, Kcenler M, Morris C. Wakeling A:Therapeutic potential of tyrosine kinase inhibitors in breast cancer.Cancer Invest 21:782-791, 2003; Baserga R: The IGF-I receptor in cancerresearch. Exp Cell Res 253:1-6, 1999; Dickson R B, Lippman M E: Growthfactors in breast cancer. Endocr Rev 16:559-589, 1995; Gross J M, Yee D:The type-1 insulin-like growth factor receptor tyrosine kinase andbreast cancer: biology and therapeutic relevance. Cancer Metastasis Rev22:327-336, 2003; and Nahta R, Hortobagyi G N, Esteva F J: Growth factorreceptors in breast cancer: potential for therapeutic intervention.Oncologist 8:5-17, 2003).

Accordingly, RTKs are targets for anti-tumor therapy. RTKs aretransmembrane proteins that typically contain an extracellular ligandbinding domain, activated by peptide hormones, and an intracellulartyrosine kinase domain. Two RTKs of demonstrated importance in breastand other cancers are the insulin-like growth factor receptor (IGF-1R)(Heinemann V: Present and future treatment of pancreatic cancer. SeminOncol 29:23-31, 2002) and c-erbB2/HER2/neu (HER2/neu) (Morin M J: Fromoncogene to drug: development of small molecule tyrosine kinaseinhibitors as anti-tumor and anti-angiogenic agents. Oncogene19:6574-6583, 2000). Based on their major role in regulating cancer cellgrowth and survival, inhibitors of these RTKs are undergoing drugdevelopment (Morin M J: From oncogene to drug: development of smallmolecule tyrosine kinase inhibitors as anti-tumor and anti-angiogenicagents. Oncogene 19:6574-6583, 2000; Bruns C J, Solorzano C C, HarbisonM T, Ozawa S, Tsan R, Fan D, Abbruzzese J, Traxler P, Buchdunger E,Radinsky R, Fidler I J: Blockade of the epidermal growth factor receptorsignaling by a novel tyrosine kinase inhibitor leads to apoptosis ofendothelial cells and therapy of human pancreatic carcinoma. Cancer Res60:2926-2935, 2000; Bruns C J, Harbison M T, Davis D W, Portera C A,Tsan R, McConkey D J, Evans D B, Abbruzzese J L, Hicklin D J, RadinskyR: Epidermal growth factor receptor blockade with C225 plus gemcitabineresults in regression of human pancreatic carcinoma growingorthotopically in nude mice by antiangiogenic mechanisms. Clin CancerRes 6:1936-1948, 2000; Blum G, Gazit A, Levitzki A: Substratecompetitive inhibitors of IGF-1 receptor kinase. Biochemistry39:15705-15712, 2000).

Signaling via the IGF-1R is important for normal cell growth anddifferentiation. In addition, the IGF-1R stimulates mitogenesis andsuppresses apoptosis of cancer cells (Lowe W L: Biological actions ofthe insulin-like growth factors. In LeRoith D (ed): Insulin-like growthfactors: molecular and cellular aspects. Boca Raton, CRC Press, 1991).Following binding of the ligand to the IGF-1R, a conformational changeinduces trans-autophosphorylation of the β-subunits on select tyrosineresidues, and subsequent activation of tyrosine kinase activity (Lowe WL: Biological actions of the insulin-like growth factors. In LeRoith D(ed): Insulin-like growth factors: molecular and cellular aspects. BocaRaton, CRC Press, 1991). Phosphorylation of several target substratesactivates divergent signaling cascades, though the anti-apoptoticeffects of the IGF-1R are primarily mediated via the Akt/PKB pathway(Kulik G, Klippel A, Weber M J: Antiapoptotic signalling by theinsulin-like growth factor I receptor, phosphatidylinositol 3-kinase,and Akt. Mol Cell Biol 17:1595-1606, 1997).

Tyrosine phosphorylation of the insulin receptor substrate (IRS) familyof proteins by the IGF-1R allows binding of the regulatory subunit ofphosphatidylinositol 3-kinase (PI3K) to the IRS proteins via SH2domains. Activated PI3K serine phosphorylates and activates the serinekinase Akt (Alessi D R, Andjelkovic M, Caudwell B, Cron P, Morrice N,Cohen P, Hemmings B A: Mechanism of activation of protein kinase B byinsulin and IGF-1. EMBO J 15:6541-6551, 1996). Akt can phosphorylate theprotein BAD, which prevents BAD from forming a pro-apoptotic complexwith Bcl-2 proteins (Virdee K, Parone P A, Tolkovsky A M:Phosphorylation of the pro-apoptotic protein BAD on serine 155, a novelsite, contributes to cell survival. Curr Biol 10:1151-1154. 2000).

Interruption of the IGF-1R signaling system, either by reducingeffective IGF-1 levels or targeting the receptor, can block growth andproliferation of cancer cells (Kahan Z, Varga J L, Schally A V, RekasiZ, Armatis P, Chatzistamou L, Czompoly T, Halmos G: Antagonists ofgrowth hormone-releasing hormone arrest the growth of MDA-MB-468estrogen-independent human breast cancers in nude mice. Breast CancerRes Treat 60:71-79, 2000: Neuenschwander S, Roberts C T, Jr., LeRoith D:Growth inhibition of MCF-7 breast cancer cells by stable expression ofan insulin-like growth factor I receptor antisense ribonucleic acid.Endocrinology 136:4298-4303, 1995; Prager D, Li H L, Asa S, Melmed S:Dominant negative inhibition of tumorigenesis in vivo by humaninsulin-like growth factor I receptor mutant. Proc Natl Acad Sci USA91:2181-2185, 1994; Weckbecker G, Tolcsvai L, Liu R, Bruns C:Preclinical studies on the anticancer activity of the somatostatinanalogue octreotide (SMS 201-995). Metabolism 41:99-103, 1992; and YeeD, Jackson J G, Kozelsky T W, Figueroa J A: Insulin-like growth factorbinding protein 1 expression inhibits insulin-like growth factor Iaction in MCF-7 breast cancer cells. Cell Growth Differ 5:73-77, 1994).While overexpression of the IGF-1R can drive transformation andmitogenesis, it is the requirement for its constitutive presence incancer cells (Rubin R, Baserga R: Insulin-like growth factor-I receptor.Its role in cell proliferation, apoptosis, and tumorigenicity. LabInvest 73:311-331, 1995) that makes this RTK an attractive target foranti-tumor therapies.

The HER2/neu (c-erbB-2) protooncogene encodes a 1,255 amino acid, 185kDa member of the class I RTK family. HER2/neu is overexpressed in20-30% of breast cancers, most commonly via gene amplification, andoverexpression is associated with poor prognosis in these patients(Slamon D J, Godolphin W, Jones L A, Holt J A, Wong S G, Keith D E,Levin W J, Stuart S G, Udove J, Ullrich A: Studies of the HER-2/neuproto-oncogene in human breast and ovarian cancer. Science 244:707-712,1989; Slamon D J, Clark G M, Wong S G, Levin W J, Ullrich A, McGuire WL: Human breast cancer: correlation of relapse and survival withamplification of the HER-2/neu oncogene. Science 235:177-182, 1987).Evidence from transgenic animal studies indicates that HER2/neuoverexpression directly contributes to transformation and tumorprogression (Bol D, Kiguchi K, Beltran L, Rupp T, Moats S, Gimenez-ContiI, Jorcano J, DiGiovanni J: Severe follicular hyperplasia andspontaneous papilloma formation in transgenic mice expressing the neuoncogene under the control of the bovine keratin 5 promoter. MolCarcinog 21:2-12, 1998; Bouchard L, Lamarre L, Tremblay P J, JolicoeurP: Stochastic appearance of mammary tumors in transgenic mice carryingthe MMTV/c-neu oncogene. Cell 57:931-936, 1989; and Lucchini F, Sacco MG, Hu N, Villa A, Brown J, Cesano L, Mangiarini L, Rindi G, Kindl S,Sessa F: Early and multifocal tumors in breast, salivary, harderian andepididymal tissues developed in MMTY-Neu transgenic mice. Cancer Lett64:203-209, 1992), and suggests that its prognostic significance arisesfrom the particularly aggressive phenotype it confers (Hynes N E, SternD F: The biology of erbB-2/neu/HER-2 and its role in cancer. BiochimBiophys Acta 1198:165-184, 1994). The efficacy of targeting HER2/neu inanti-cancer therapy has been demonstrated by the clinical use of anantibody to HER2/neu to treat certain patients with breast cancer(Albanell J, Baselga J: Trastuzumab, a humanized anti-HER2 monoclonalantibody, for the treatment of breast cancer. Drugs Today (Bare)35:931-946, 1999).

Nordihydroguaiaretic acid (NDGA) is a phenolic compound that wasidentified as a major component of a tea made from resinous extracts ofthe creosote bush Larrea divaricatta. It has been used for centuries byNative North Americans as a remedy for diverse illnesses, includingtumors (Duisberg P C: Desert Plant Utilization. Texas J Sci 4:269, 1952;Hawthorn P: Medicinal uses of plants of Nevada used by Indians. ContrFlora Nevada 45:1-139, 1957). NDGA has been reported to inhibit thegrowth of various tumors both in vitro and in animals (Wilson D E,DiGianfilippo A, Ondrey F G, Anderson K M, Harris J E: Effect ofnordihydroguaiaretic acid on cultured rat and human glioma cellproliferation. J Neurosurg 71:551-557, 1989; Avis T M, Jett M, Boyle T,Vos M D, Moody T, Treston A M, Martinez A, Mulshine J L: Growth controlof lung cancer by interruption of 5-lipoxygenase-mediated growth factorsignaling. J Clin Invest 97:806-813, 1996; Rose D P, Connolly J M:Effects of fatty acids and inhibitors of eicosanoid synthesis on thegrowth of a human breast cancer cell line in culture. Cancer Res50:7139-7144, 1990; and Shimakura S. Boland C R: Eicosanoid productionby the human gastric cancer cell line AGS and its relation to cellgrowth. Cancer Res 52:1744-1749, 1992). NDGA also has been reported toinduce apoptosis in a variety of cell lines (Ding X Z, Kuszynski C A, ElMetwally T H, Adrian T E: Lipoxygenase inhibition induced apoptosis,morphological changes, and carbonic anhydrase expression in humanpancreatic cancer cells. Biochem Biophys Res Commun 266:392-399, 1999;La E, Kern J C, Atarod E B, Kehrer J P: Fatty acid release and oxidationare factors in lipoxygenase inhibitor-induced apoptosis. Toxicol Lett138:193-203, 2003; Seufferlein T, Seckl M J, Schwarz E, Beil M, WichertG, Baust H, Luhrs H, Schmid R M, Adler G: Mechanisms ofnordihydroguaiaretic acid-induced growth inhibition and apoptosis inhuman cancer cells. Br J Cancer 86:1188-1196, 2002; Tong W G, Ding X Z,Witt R C, Adrian T E: Lipoxygenase inhibitors attenuate growth of humanpancreatic cancer xenografts and induce apoptosis through themitochondrial pathway. Mol Cancer Ther 1:929-935, 2002; and Tong W G,Ding X Z, Adrian T E: The mechanisms of lipoxygenase inhibitor-inducedapoptosis in human breast cancer cells. Biochem Biophys Res Commun296:942-948, 2002). Still, the mechanism of this anti-cancer effect ofNDGA is not well understood. It has been reported that NDGA inhibits thetyrosine kinase activity of the platelet-derived growth factor receptor(PDGFR), but not the epidermal growth factor receptor (EGFR), in cellsand in vitro (Domin J, Higgins T, Rozengurt E: Preferential inhibitionof platelet-derived growth factor-stimulated DNA synthesis and proteintyrosine phosphorylation by nordihydroguaiaretic acid. J Biol Chem269:8260-8267, 1994). While one report suggests that NDGA is inactiveagainst the IGF-1R (Seufferlein T, Seckl M J, Schwarz E, Bell M, WichertG, Baust H, Luhrs H, Schmid R M, Adler G: Mechanisms ofnordihydroguaiaretic acid-induced growth inhibition and apoptosis inhuman cancer cells. Br J Cancer 86:1188-1196, 2002), a compound with avery high degree of structural homology to NDGA has been described as apotent inhibitor of this receptor(Blum G, Gazit A, Levitzki A: Substratecompetitive inhibitors of IGF-1 receptor kinase. Biochemistry39:15705-15712, 2000: Blum G, Gazit A, Levitzki A: Development of newinsulin-like growth factor-1 receptor kinase inhibitors using catecholmimics. J Biol Chem 278:40442-40454, 2003). The effects of NDGA on theHER2/neu receptor, which also plays a critical role in breast cancer,have not been explored. We have now found that NDGA antagonizes theactivation of both the IGF-1 and HER2/neu receptors, inhibits thecellular anti-apoptotic signaling pathway of the IGF-1R, and inhibitsthe growth of breast cancer cells both in vitro and in vivo.

There is a need for therapeutic cancer treatments that block thetyrosine kinase receptors with lower dosages of these powerful drugs toreduce side effects. The present invention addresses this and otherrelated needs.

SUMMARY OF THE INVENTION

Treatments for breast and pancreatic cancer are disclosed. The malignantcells are brought into contact with NDGA and diarylurea 21834, eithersingly or in combination with other compounds, such as doxorubicin andHerceptin.

Nordihydroguaiaretic acid (NDGA) is a phenolic compound isolated fromthe creosote bush Larrea divaricatta that has anti-cancer activitiesboth in vitro and in vivo. These anti-cancer properties in breast cancercells are created by the ability of NDGA to directly inhibit thefunction of two receptor tyrosine kinases (RTKs), the insulin-likegrowth factor receptor (IGF-1R) and the c-erbB2/HER2/neu (HER2/neu)receptor. In MCF-7 human breast cancer cells, low micromolarconcentrations of NDGA inhibited activation of the IGF-1R, anddownstream phosphorylation of both the Akt/PKB serine kinase and thepro-apoptotic protein BAD.

In mouse MCNeuA cells, NDGA also inhibited ligand independentphosphorylation of HER2/neu. This inhibitory effect in cells is due to adirect action on these receptors. The IGF-1-stimulated tyrosine kinaseactivity of isolated IGF-1R is inhibited by NDGA at 10 μM or less. Acomposition of NDGA is also effective at inhibiting autophosphorylationof isolated HER2/neu receptor at similar concentrations. In addition,NDGA inhibits IGF-1 specific growth of cultured breast cancer cells withan IC50 of approximately 30 μM. Treatment with NDGA (intraperitonealinjection 3 times per week) also decreases the activity of the IGF-1Rand the HER2/neu receptor in MCNeuA cells implanted into mice.Inhibition of RTK activity is associated with decreased growth rates ofMCNeuA cells in vivo. Accordingly, the anti-breast cancer properties ofNDGA are related to the inhibition of two important RTKs and as suchformulations of RTK inhibitors provide a means of treating breastcancer.

One aspect of the invention comprises methods for using NDGA in thetreatment of breast cancer.

A further aspect of the invention is methods for treating breast cancerwith a combination of NDGA and Doxorubicin. This formulation provides anunexpected synergistic effect in combination at low concentrationscompared to individual dosages. When given in combination, lower dosagesmay be used to achieve a greater effect, which has the additionalbenefit of decreasing side-effects compared to the individual drugsgiven at higher dosages.

Another aspect of the invention is methods for using Diarylurea 21834(DAU) alone or in various combinations with other compounds for thetreatment of breast cancer.

One aspect of the invention comprises methods for using NDGA in thetreatment of pancreatic cancer.

A further aspect of the invention is methods for treating pancreaticcancer with a combination of NDGA and Doxorubicin. This formulationprovides an unexpected synergistic effect in combination at lowconcentrations compared to individual dosages. When given incombination, lower dosages may be used to achieve a greater effect,which has the additional benefit of decreasing side-effects compared tothe individual drugs given at higher dosages.

Another aspect of the invention is methods for using Diarylurea 21834(DAU) alone or in various combinations with other compounds for thetreatment of pancreatic.

As aspect of the invention is treating a breast cancer patient byadministering tamoxifen to the patient; determining that the patient isnot sufficiently responsive to tamoxifen; and treating the patient witha combination of tamoxifen and NDGA.

Another aspect of the invention is diagnosing a cancer patient as havingestrogen receptor (ER) positive MCF-7 cells that overexpress HER2(MCF-7/HER2-18) and administering to the patient a therapeuticallyeffective amount of a combination of tamoxifen and NDGA.

Another aspect of the invention is a formulation manufactured for thetreatment of breast cancer specifically where the cancer has been shownto be resistant to tamoxifen, wherein the formulation comprises atherapeutically effective amount of a combination of tamoxifen and NDGA.

Another aspect of the invention is a method of treatment wherein apatient is treated with tamoxifen and found to be insufficientlyresponsive which treatment is followed by administration of bothtamoxifen and NDGA.

Still yet another aspect of the invention is a kit comprised oftamoxifen, NDGA, and instructions with respect to the treatment ofpatients having estrogen receptor (ER) positive MCF-7 cells thatoverexpress HER2 (MCF-7/HER2-18).

These and other aspects of the invention will become apparent to thosepersons skilled in the art upon reading the details of the formulationsand methods as more fully described below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the chemical structure of nordihydroguaiaretic acid (NDGA).

FIG. 2 shows the chemical structure of diarylurea 21834 (DAU).

FIG. 3 is a graph showing the results of using NDGA to inhibit thegrowth of pancreatic cancer cells in vitro.

FIG. 4A is a graph showing the effectiveness of NDGA in inhibiting IGF-1activation in pancreatic cancer cells. FIG. 4B is a graph showing theeffectiveness of NDGA in inhibiting Her2/neu activation in pancreaticcancer cells.

FIG. 5 is a graph showing that DAU inhibits tyrosine kinase activationin Panc 1 pancreatic cancer cells.

FIG. 6A is a schematic representation of an isobologram and FIG. 6B is aschematic representation of an isobologram analysis for DOX and NDGA onSKBR-3 cells.

FIG. 7 is a bar graph showing the affect of different concentrations ofNDGA on prostate cancer cells.

FIG. 8 is an image of a Western Blot obtained by incubating differentconcentrations of NDGA with a peptide corresponding to the kinase domainof IGF-1R.

FIG. 9 is a bar graph showing the results of IGF-1R incubated withvarious concentrations of NDGA prior to addition of IGF-1 whereinresults were determined by ELISA employing an anti-phosphotyrosineantibody to readout.

FIG. 10 is an image of a Western Blot wherein HER2/neu recepetors wereincubated with ATP alone or with NDGA.

FIG. 11A is a bar graph and FIG. 11B is two images of Western Blotsshowing results where MCF-7 breast cancer cells were incubated withvarious concentrations of NDGA.

FIG. 12 is an image of a Western Blot showing results wherein MCNeuAbreast cancer cells were incubated with varying concentrations of NDGA.

FIG. 13A is a bar graph showing results of incubating MCF-7 cells withvarying concentrations of NDGA and FIG. 13B is a bar graph of resultsshowing results wherein such cells were incubated in serum or aconcentration of 10 nM IGF-1.

FIG. 14 is a bar graph showing results wherein NDGA was administeredover a period of 21 days in vivo to MCNeuA tumors.

FIG. 15 is a graph showing the results of MDNeuA cells of a tumor beingtreated over a period of days with NDGA.

FIGS. 16A and 16B are each bar graphs showing the affect of DMSO andNDGA on neuroblastoma cells.

FIGS. 17A and 17B are each bar graphs showing the affect of DMSO andfour different concentrations of NDGA on neuroblastoma cells.

FIGS. 18A and 18B are each bar graphs showing the affect of DMSO andthree different concentrations of NDGA on neuroblastoma cells.

FIGS. 19A and 19B are each images of Western Blot analysis showingresults obtained with DMSO and three different concentrations of NDGA.

FIG. 20A is an image of a Western Blot showing the affects of DMSO andthree different concentrations of NDGA. FIG. 20B is an image of aWestern Blot showing the affects of DMSO and NDGA. FIG. 20C is a bargraph showing the results obtained using DMSO and NDGA in threedifferent concentrations.

FIGS. 21A and 21B are each bar graphs showing the affects of DMSO andNDGA. FIG. 21C shows the affects of DMSO and NDGA used to treat mice byinjection.

FIG. 22 shows gel images which show the expression of IGF-IR and HER2 inMCF-7/neo and MCF-7/HER2-18 cells. The gels were created using celllysates which were separated by SDS-PAGE, transferred to nitrocellulosemembranes, and probed with antibodies specific for IGF-1R and HER2. Twoconcentrations of total protein 10 μg and 14 μg were analyzed to confirmthe linearity of the assay.

FIGS. 23A and 23B are each graphs which show the effects of gefitiniband NDGA on the growth of MCF-7/neo and MCF-7/HER2-18 cells. Cells weregrown in the presence of various concentrations of gefitinib (23A) orNDGA (23B) for 6 days. Cell growth was assessed with a CyQuant cellproliferation assay. The results are expressed as mean±SEM of triplicatewells and are representative of three separate experiments.

FIGS. 24A and 24B are each graphs which show the effects of gefitiniband NDGA on IGF-IR and HER2 phosphorylation in MCF-7/HER2-18 cells.Cells treated with various concentrations of gefitinib (A) or NDGA (B)were lysed and assayed for IGF-1R and HER2 phosphorylation by ELISA. Forthe IGF-1R ELISA, cells were stimulated with 3 nM IGF-I for 10 min. Forthe HER2 ELISA, cells were not stimulated. The results are expressed asmean±SEM of triplicate wells and are representative of three separateexperiments. The effects of NDGA were confirmed by Western blot (panelB, inset) where phosphorylated IGF-IR and HER2 were detected withphospho-specific antibodies to pIGF-IR and pHER2, respectively.

FIG. 25 shows gel images which show the effect of NDGA on Akt/PKBphosphorylation. MCF-7/HER2-18 cells were incubated in the presence orabsence of 3 nM IGF-1 for 10 min, with or without NDGA treatment. Cellslysates were prepared and separated by SDS-PAGE, transferred tonitrocellulose membranes, and probed with an anti-pAkt antibody.

FIGS. 26A, 26B, 26C and 26D are graphs which show the effects ofcombined treatment with tamoxifen and NDGA on the growth of MCF-7/neoand MCF-7/HER2-18 cells. MCF-7/neo (panels 26A and 26C) andMCF-7/HER2-18 (panels 26B and 26D) cells were incubated for 6 days with100 nM tamoxifen, in the presence or absence of various concentrationsof NDGA and cell growth was assessed with a CyQuant assay. Cellproliferation was expressed as a percentage of untreated control cells(mean±SEM) (panels 26A and 26B). Panels 26C and 26D express the resultsas percent growth inhibition.

FIG. 27 is a graph which shows results where human breast cancer MCF-7cells were grown for 5 days with medium plus 10% fetal calf serum andtreated with the indicated concentrations of valproic acid (VAPA),nordihydroguaiaretic acid (NDGA), or rapamycin (RAPA). At the end of 5days the cultures (in a 96 well plate) were assayed for total nucleicacid with a CyQuant dye based assay. Shown is the OD in this assay.

DETAILED DESCRIPTION OF THE INVENTION

Before the present compositions for and methods of treating cancer aredescribed, it is to be understood that this invention is not limited toparticular compositions and methods described, as such may, of course,vary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to be limiting, since the scope of the present invention willbe limited only by the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, potential andpreferred methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “acancer cell” includes a plurality of such cancer cells and reference to“the methods of administration” includes reference to one or moremethods and equivalents thereof known to those skilled in the art, andso forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

Definitions

The term “nordihydroguaiaretic acid” is also referred to as “NDGA” andis the compound shown within the structure of FIG. 1 and see U.S. Pat.No. 2,644,822 incorporated here to disclosed NDGA as well as relatedcompounds and their method of manufacture. It is pointed out thatpharmaceutically acceptable salts and amines of the acid may be formedduring use and are considered to be encompassed by the term unlessspecifically indicated otherwise.

The term tyrosine kinase receptor blocker and inhibitor of tyrosinekinase are used interchangeably to describe compounds which selectivelyand specifically bind to tyrosine kinase receptors. The bindingpreferably has an antagonist effect. Such compounds include compoundssuch as Her2 inhibitors, doxorubicine and Herceptin™.

The terms “treatment,” “treating,” and the like are used herein togenerally mean obtaining a desired pharmacological and/or physiologicaleffect. The effect may be prophylactic in tee ins of completely orpartially preventing a disease or symptom thereof and/or may betherapeutic in terms of partially or completely curing a disease and/oradverse effect attributed to the disease. In general, methods of theinvention involve treating diseases referred to as cancer and may beapplied to a variety of different types of cancer by utilizingcombinations of compounds such as tyrosine kinase receptor inhibitorswhich are known to bind to the receptor site. “Treatment” as used hereincovers any treatment of such a disease in a mammal, particularly ahuman, and includes:

(a) preventing and/or diagnosing the disease in a subject which may bepredisposed to the disease which has not yet been diagnosed as havingit;

(b) inhibiting the disease, i.e. arresting its development; and/or

(c) relieving the disease, i.e. causing regression of the disease.

The invention is directed towards treating patients with cancer and isparticular directed towards treating particular types of cancer whichare not generally treatable with normal surgical methods. Morespecifically, “treatment” is intended, in preferred circumstances, tomean providing a therapeutically detectable and beneficial effect on apatient suffering from cancer.

Formulations and Methods

Formulations of the invention combine compounds and excipients to obtaindesirable results with respect to the biochemical inhibition of certainreceptors. The compound such as nordihydroguaiaretic acid (NDGA), IFG-1inhibitors and recombinant Her2 inhibitors as well as doxorubicine andHerceptin™ can be used in a pharmaceutically acceptable excipientcarrier in various combinations. The combinations of the inventionobtain a synergistic effect. This synergistic effect is specificallydefined in connection with the present invention. Those skilled in theart will understand that the use of compound A to inhibit receptor Xcannot be increased beyond certain points simply by adding more ofcompound A. At some point the effect of compound A is not increased byadding the amount or the increase is not practical in view of the toxiceffects. Thus, the combination of “compound A” and “compound B” may besynergistic in blocking receptor “X” even when the combination of “A”and “B” is not additive in terms of blocking receptor “X”. Obtaining, amodest increase in the blockage of receptors without an increase inadverse effects or even with an acceptable level of adverse effects maybe all that is necessary to effectively treat a given cancer.

Certain compounds may be used by themselves in particular dosage amountsand treatment regimes. For example, NDGA may be used in the treatment ofpancreatic cancer. FIG. 3 shows the results of NDGA inhibiting thegrowth of pancreatic cancer cells in vitro. FIGS. 4A and 4B show theeffectiveness of NDGA in inhibiting certain receptors. Specifically,FIG. 4A shows NDGA in micrograms per milliliter inhibiting Her2/NEUactivation in pancreatic cancer cells. FIG. 4B shows NDGA being used inmicrograms per milliliter inhibiting IGF-1 activation in pancreaticcancer cells.

FIG. 5 is a graph showing that DAU inhibits tyrosine kinase activationin Panc 1 pancreatic cancer cells.

FIG. 6A is a schematic representation of an isobologram. The envelope ofadditivity, surrounded by Mode I (solid line) and Mode II (dotted lines)isobologram lines, is constructed from the dose-response curves of twodrugs (A and B). The data points Pa, Pb, Pc and Pd, obtained from thecombination dose-response curves of A and B, would classify the two-druginteractions as supra-additive (synergistic), additive, sub-additive andmutually protective, respectively. FIG. 6B is an isobologram analysisfor DOX and NDGA on SKBR-3 cells. At low concentrations, the twocompounds act synergistically.

When the combinations of drugs are used in accordance with the inventionimproved results of some manner are expected with respect to inhibitingthe growth of cancer cells. FIG. 6A is a graph which schematicallyrepresents a basic concept behind an aspect of the invention. FIG. 6Ashows an envelope of additivity for two drugs “A” and “B”. As indicatedin the graph the amount of drug: “B” increases in an upward directionalong the “X” axis and the amount of drug “A” increases to the rightalong the “Y” axis. Using less drug is generally more desirable when thesame amount of the drug can obtain the desired effect. Thus, the solidline and the area below the solid line represents a supra-additiveeffect.

FIG. 7 is a bar graph showing the affect of NDGA on PC3 prostate cancercell growth wherein the cells were plated at 10⁵ per well in the absenceand presence of NDGA for four days with the results shown as themean±SD4 triplicate determinations.

The present invention shows that NDGA is a direct inhibitor of both theIGF-1R and the HER2/neu receptor in isolated receptor preparations,cultured breast cancer cells, and tumors in vivo. Inhibition of theseRTKs was accompanied by a reduction in cell growth both in cell cultureand in vivo. The present invention demonstrates studies indicate thatinhibition of RTKs has an important anti-tumor effect of NDGA.

The IGF-1R is an essential component of the transformation process andan attractive target for anti-cancer agents (Gross J M, Yee D: Thetype-1 insulin-like growth factor receptor tyrosine kinase and breastcancer: biology and therapeutic relevance. Cancer Metastasis Rev22:327-336, 2003). Cells in tissue culture that lack the IGF-1R can notbe transformed (Baserga R: Oncogenes and the strategy of growth factors.Cell 79:927-930, 1994). Following transformation, overexpression ofIGF-1R is observed in many cell types (Rubin R, Baserga R: Insulin-likegrowth factor-I receptor. Its role in cell proliferation, apoptosis, andtumorigenicity. Lab Invest 73:311-331, 1995). In vivo, in primary breasttumors, the IGF-1R is overexpressed and hyperphosphorylated (Arteaga CL, Kitten L J, Coronado E B, Jacobs S, Kull F C, Jr., Allred D C,Osborne C K: Blockade of the type I somatomedin receptor inhibits growthof human breast cancer cells in athymic mice. J Clin Invest84:1418-1423, 1989). In vitro, in a variety of breast cancer cell lines,we have found that the IGF-1R is overexpressed (Sciacca L, Costantino A,Pandini G, Mineo R, Frasca F, Scalia P, Sbraccia P, Goldfine I D,Vigneri R, Belfiore A: Insulin receptor activation by IGF-II in breastcancers: evidence for a new autocrine/paracrine mechanism. Oncogene18:2471-2479, 1999). In addition, there is strong evidence linkinghyperactivation of the IGF-1R with the early stages of breast cancer(Baserga R: The IGF-I receptor in cancer research. Exp Cell Res 253:1-6,1999; Khandwala H M, McCutcheon I E, Flyvbjerg A, Friend K E: Theeffects of insulin-like growth factors on tumorigenesis and neoplasticgrowth. Endocr Rev 21:215-244, 2000; and Suiinacz E: Function of theIGF-I receptor in breast cancer. J Mammary Gland Biol Neoplasia5:95-105, 2000). Anti-proliferative effects against cultured breastcancer cells have been observed by employing antisense IGF-1R(Neuenschwander S, Roberts C T, Jr., LeRoith D: Growth inhibition ofMCF-7 breast cancer cells by stable expression of an insulin-like growthfactor I receptor antisense ribonucleic acid. Endocrinology136:4298-4303, 1995), monoclonal antibodies (Sachdev D, Li S L, HartellJ S, Fujita-Yamaguchi Y, Miller J S, Yee D: A chimeric humanizedsingle-chain antibody against the type I insulin-like growth factor(IGF) receptor renders breast cancer cells refractory to the mitogeniceffects of IGF-I. Cancer Res 63:627-635, 2003; Arteaga C L, Osborne C K:Growth inhibition of human breast cancer cells in vitro with an antibodyagainst the type I somatomedin receptor. Cancer Res 49:6237-6241, 1989),transfection with a dominant negative IGF-1R (Prager D, Li H L, Asa S,Melmed S: Dominant negative inhibition of tumorigenesis in vivo by humaninsulin-like growth factor I receptor mutant. Proc Natl Acad Sci USA91:2181-2185, 1994), or small-molecule catechol mimics (Blum G, Gazit A,Levitzki A: Development of new insulin-like growth factor-1 receptorkinase inhibitors using catechol mimics. J Biol Chem 278:40442-40454,2003). Expressing dominant negative IGF-1R in breast cancer cells alsoinhibits tumor growth in vivo (Prager D, Li H L, Asa S, Melmed S:Dominant negative inhibition of tumorigenesis in vivo by humaninsulin-like growth factor I receptor mutant. Proc Natl Acad Sci USA91:2181-2185, 1994).

HER2/neu has also emerged as an important target in breast cancertherapeutics. HER2/neu overexpression occurs in approximately half ofDCIS cases, and 85-100% of high-grade, comedo-type DCIS, the lesionsassociated with the highest risk of progression (Wu Y, Tewari M, Cui S,Rubin R: Activation of the insulin-like growth factor-I receptorinhibits tumor necrosis factor-induced cell death. J Cell Physiol168:499-509, 1996; Prisco M, Hongo A, Rizzo M G, Sacchi A, Baserga R:The insulin-like growth factor I receptor as a physiologically relevanttarget of p53 in apoptosis caused by interleukin-3 withdrawal. Mol CellBiol 17:1084-1092, 1997; and Tanno S, Tanno S, Mitsuuchi Y, Altomare DA, Xiao G H, Testa J R: AKT activation up-regulates insulin-like growthfactor I receptor expression and promotes invasiveness of humanpancreatic cancer cells. Cancer Res 61:589-593, 2001). The effectivenessof a monoclonal antibody that inhibits HER2/neu indicates the potentialof this RTK as an anti-cancer target, although no small-moleculecompounds specifically targeting HER2/neu with efficacy in vivo havebeen reported.

NDGA has previously been shown to reduce growth and induce apoptosis ina wide variety of cell lines including breast, lung, pancreas cancers(Moody T W, Leyton J, Martinez A, Hong S, Malkinson A, Mulshine J L:Lipoxygenase inhibitors prevent lung carcinogenesis and inhibitnon-small cell lung cancer growth. Exp Lung Res 24:617-628, 1998; ChenX, Li N, Wang S, Hong J, Fang M, Yousselfson J, Yang P, Newman R A,Lubet R A, Yang C S: Aberrant arachidonic acid metabolism in esophagealadenocarcinogenesis, and the effects of sulindac, nordihydroguaiareticacid, and alpha-difluoromethylomithine on tumorigenesis in a ratsurgical model. Carcinogenesis 23:2095-2102, 2002; Hausott B, Greger H,Marian B: Naturally occurring lignans efficiently induce apoptosis incolorectal tumor cells. J Cancer Res Clin Oncol 129:569-576, 2003;Wagenknecht B, Schulz J B, Gulbins E, Weller M: Crm-A, bcl-2 and NDGAinhibit CD95L-induced apoptosis of malignant glioma cells at the levelof caspase 8 processing. Cell Death Differ 5:894-900, 1998;Schultze-Mosgau M H, Dale I L, Gant T W, Chipman J K, Kerr D J, GescherA: Regulation of c-fos transcription by chemopreventive isoflavonoidsand lignans in MDA-MB-468 breast cancer cells. Eur J Cancer34:1425-1431, 1998), but its anti-tumor action has not been defined. Theprincipal finding of the present study is therefore the attribution ofthe described anti-cancer actions to the ability of NDGA to inhibitcellular RTK activity. The ability of NDGA to inhibit growth in normalculture conditions is consistent with studies employing specificinhibition of IGF-1R signaling by antibody or molecular techniques(Neuenschwander S, Roberts C T, Jr., LeRoith D: Growth inhibition ofMCF-7 breast cancer cells by stable expression of an insulin-like growthfactor I receptor antisense ribonucleic acid. Endocrinology136:4298-4303, 1995; Arteaga C L, Osborne C K: Growth inhibition ofhuman breast cancer cells in vitro with an antibody against the type Isomatomedin receptor. Cancer Res 49:6237-6241, 1989). In addition, wehave demonstrated that NDGA was more effective at inhibiting growthstimulated solely by IGF-1 than at inhibiting growth stimulated by thecomplex milieu provided by fetal calf serum, or growth in the absence ofall exogenous growth factors. These results suggest that inhibition ofthe IGF-1R comprises a major component of the anti-mitogenic effects ofNDGA in cell culture. The findings that NDGA inhibition of the IGF-1Rproduces a subsequent reduction in phosphorylation of the serine kinaseAkt/PKB, and of the pro-apoptotic protein BAD, provide evidence that thedescribed apoptotic effects of NDGA treatment result directly from aninhibition of the cell survival pathway regulated by the IGF-1R.

We also found that NDGA was able to reduce phosphorylation of the IGF-1Rand HER2/neu in tumors in vivo. This action against these RTKs wasassociated with significant reductions in tumor cell growth. While otherstudies have demonstrated the effectiveness of NDGA against xenograftmodels of pancreatic and non-small cell lung cancer (Tong W G, Ding X Z,Witt R C, Adrian T E: Lipoxygenase inhibitors attenuate growth of humanpancreatic cancer xenografts and induce apoptosis through themitochondrial pathway. Mol Cancer Ther 1:929-935, 2002; Moody T W,Leyton J, Martinez A, Hong S, Malkinson A, Mulshine J L: Lipoxygenaseinhibitors prevent lung carcinogenesis and inhibit non-small cell lungcancer growth. Exp Lung Res 24:617-628, 1998), in the present study wewere able to demonstrate that chronic treatment of tumor-bearing miceresulted in a reduction in the normal, physiological activation state ofboth IGF-1 and c-erbB2/HER2/neu receptors. This effect was observed 16hours after treatment, and is unlikely to be the result of an acuteeffect of the previous injection. Thus, we believe that theRTK-inhibitory actions of NDGA contribute greatly to its in vivoanti-cancer properties as well.

The terminal half-life for a single injection of NDGA is reported to be135 minutes (Lambert J D, Meyers R O, Timmermann B N, Dorr R T:Pharmacokinetic analysis by high-performance liquid chromatography ofintravenous nordihydroguaiaretic acid in the mouse. J Chromatogr BBiomed Sci Appl 754:85-90, 2001), although the half-life in tissues aswell as the potency of potential metabolites of NDGA are unknown.Although extracts of the creosote bush have demonstrated toxic effects(Arteaga S, Andrade-Cetto A, Cardenas R: Larrea tridentata (Creosotebush), an abundant plant of Mexican and US-American deserts and itsmetabolite nordihydroguaiaretic acid. J Ethnopharmacol 98:231-239,2005), Pure NDGA itself has minimal toxicity, has passed FDA approvedpre-clinical trials and is available for administration to humans (G.Kelly, Insmed Inc., Glen Allen, Va., personal communication).

The mechanism whereby NDGA inhibits RTK activity has not yet beenelucidated. Most small-molecule RTK inhibitors are competitiveinhibitors of ATP binding (Morin M J: From oncogene to drug: developmentof small molecule tyrosine kinase inhibitors as anti-tumor andanti-angiogenic agents. Oncogene 19:6574-6583, 2000), as is a recentlyreported IGF-1R-specific inhibitor (Mitsiades C S, Mitsiades N S,McMullan C J, Poulaki V, Shringarpure R, Akiyama M, Hideshima T, ChauhanD, Joseph M, Libermann T A, Garcia-Echeverria C, Pearson M A, Hofmann F,Anderson K C, Kung A L: Inhibition of the insulin-like growth factorreceptor-1 tyrosine kinase activity as a therapeutic strategy formultiple myeloma, other hematologic malignancies, and solid tumors.Cancer Cell 5:221-230, 2004; Garcia-Echeverria C, Pearson M A, Marti A,Meyer T, Mestan J, Zimmermann J, Gao J, Brueggen J, Capraro H G, CozensR, Evans D B, Fabbro D, Furet P, Porta D G, Liebetanz J, Martiny-BaronG, Ruetz S, Hofmann F: In vivo antitumor activity of NVP-AEW541-A novel,potent, and selective inhibitor of the IGF-IR kinase. Cancer Cell5:231-239, 2004). However, NDGA does not share general structuralhomology with ATP analogs. Interestingly, Blum et al. (Blum G, Gazit A,Levitzki A: Substrate competitive inhibitors of IGF-1 receptor kinase.Biochemistry 39:15705-15712, 2000: Blum G, Gazit A, Levitzki A:Development of new insulin-like growth factor-1 receptor kinaseinhibitors using catechol mimics. J Biol Chem 278:40442-40454, 2003)have investigated a compound that appears to inhibit IGF-1R signaling bycompeting with the autophosphorylation sites on the β-subunit of theIGF-1R. This compound was effective in inhibiting the growth of breastcancer cells in culture. Interestingly, the di-catechol structureinvestigated by this group shares considerable structural homology withNDGA, differing only in length of the carbon chain linking the twocatechol rings. Modeling data generated by these investigators with theclosely related IR suggest that their lead compound competes for bindingwith the tyrosine substrates at the kinase active site (Blum G, Gazit A,Levitzki A: Substrate competitive inhibitors of IGF-1 receptor kinase.Biochemistry 39:15705-15712, 2000). It is unclear however whether NDGAinteracts with a homologous site on either IGF-1R or HER2/neu. Theability of NDGA to inhibit autophosphorylation of intrinsically activeHER2/neu suggests that this compound acts directly on the enzymaticcomponents of the receptors and not by interfering with either ligandbinding or binding-dependent conformational changes. Thus, NDGA mayrepresent a potential new class of agents for the treatment of breastand other cancers where the IGF-1R and/or HER2/neu play a role in theoncogenic process.

Treating Neuroblastoma

Neuroblastoma is a common pediatric malignancy that metastasizes to theliver, bone, and other organs, and is often resistant to availabletreatments. Insulin-like growth factors (IGFs) stimulate neuroblastomagrowth, survival, and motility, and are expressed by neuroblastoma cellsand the tissues they invade. Administration of formulations of theinvention disrupt the effects of IGFs on neuroblastoma tumorigenesis andthereby slow disease progression. Nordihydroguaiaretic acid (NDGA), aphenolic compound isolated from the creosote bush (Larrea divaricata),has anti-tumor properties against a number of malignancies. NDGAinhibits the phosphorylation and activation of the Her2/neu and IGF-Ireceptors (IGF-IR). The present invention shows that NDGA inhibitsIGF-I-mediated activation of the IGF-IR in human neuroblastoma celllines. NDGA inhibits neuroblastoma growth and disrupts activation of ERKand Akt signaling pathways induced by IGF-I. NDGA induces apoptosis athigher doses, causing IGF-I-resistant activation of caspase-3 and alarge increase in the fraction of sub-G₀ cells. NDGA inhibits the growthof xenografted human neuroblastoma tumors in nude mice by 50%. Theresults provided show that small molecules that prevent activation ofthe IGF-IR, such as NDGA, are useful in the treatment of neuroblastoma.

Neuroblastoma affects an estimated 1 in 7000 children under age 15(Carlsen N L. Neuroblastoma: epidemiology and pattern of regression.Problems in interpreting results of mass screening. Am J Pediatr HematolOncol 1992;14:103-110), making it the second most common solid tumor inchildren. Neuroblastoma tumors are believed to arise from neural crestcells in the adrenal gland and spinal ganglia. Neuroblastoma oftenregresses spontaneously in children under 1 year of age, butneuroblastoma in older children is difficult to treat with conventionalradiation and chemical therapies (Philip T. Overview of currenttreatment of neuroblastoma. Am J Pediatr Hematol Oncol 1992;14:97-102).Metastasis to bone, meninges, the liver, and other organs contributes tothe difficulty in eliminating the disease.

The development of effective treatments for neuroblastoma is hampered byan incomplete understanding of the factors that lead to neuroblastomatumorigenesis, although several key abnormalities are associated with asinificant subset of aggressive tumors. Although several chromosomalabnormalities have been described, amplification of MYCN is still thebest understood genetic abnormality and is associated with advanceddisease.

While a primary defect in growth factor signaling has not been observedin neuroblastoma, growth factor responsiveness is believed to supporttumor growth, survival, and invasiveness. Thus, therapeutic approachesthat disrupt growth factor signaling may have an impact on diseaseprogression. Recently, nordihydroguaiaretic acid (NDGA), a naturallyoccurring compound isolated from creosote (Larrea divaricata), was foundto inhibit the activation of partially purified insulin-like growthfactor I (IGF-I) and her2/neu receptor tyrosine kinases.

NDGA has been extensively studied as an inhibitor of arachidonic acidmetablolism, where it blocks lipoxygenase activity, but its ability toinhibit receptor tyrosine kinase activation was previouslyunappreciated. In breast cancer cells, NDGA inhibited ligand activationof the IGF-I and her2/neu receptors and subsequent activation ofsignaling intermediates downstream of these receptors. Both the in vitroand in vivo growth of breast cancer cells is inhibited by NDGA,potentially via its ability to suppress responsiveness to growthfactors.

IGF-I and II are peptide growth factors that regulate cell mitogenesisand survival. IGFs bind to the tyrosine kinase IGF-I receptor (IGF-IR),causing receptor autophosphorylation that initiates the mitogenactivated protein kinase (MAPK) and phosphatidylinositol 3-kinase(PI-3K) signaling pathways. MAPK regulates mitogenesis (De Meyts P,Wallach B, Christoffersen C T, et al. The insulin-like growth factor-Ireceptor. Structure, ligand-binding mechanism and signal transduction.Horm Res 1994:42:152-169), while PI-3K activates targets that impactapoptosis, such as Akt (Fresno Vara J A, Casado E, de Castro J, Cejas P,Belda-Iniesta and Gonzalez-Baron M. PI3K/Akt signaling pathway andcancer. Cancer Treat Rev 2004;30:193-204).

IGFs promote neuroblastoma tumorigencity by stimulating proliferation,inhibiting apoptosis, and stimulating motility. IGFs are expressed inall neuroblastoma tumor stages and in neuroblastoma tumor lines (MartinD M, Yee D, Carlson R O and Feldman E L. Gene expression of theinsulin-like growth factors and their receptors in human neuroblastomacell lines. Brain Res Mol Brain Res 1992;15:241-246), and can act aseither autocrine or paracrine mitogens (Martin D M and Feldman E L.Regulation of insulin-like growth factor-II expression and its role inautocrine growth of human neuroblastoma cells. J Cell Physiol1993;155:290-300). IGF-I and IGF-IR expression prevent neuroblastomacells from undergoing apoptosis (Singleton J R, Dixit V M and Feldman EL. Type I insulin-like growth factor receptor activation regulatesapoptotic proteins. J Biol Chem 1996;271:31791-31794) by regulating theactivity of caspases and Bcl proteins. IGFs also regulate the metastaticcapabilities of neuroblastoma cells by stimulating actin polymerization,lamellipodium extension, and motility.

The present invention is based, in part, on an understanding of theability of NDGA to inhibit growth and IGF-IR-related signaling events inbreast cancer. The present invention shows the anti-tumor effects ofNDGA in three human neuroblastoma cell lines. Results provided herequantify IGF-I- and serum-dependent growth of neuroblastoma cellstreated with NDGA, and characterize IGF-I-dependent phosphorylation ofIGF-IR, extracellular regulated kinases (ERKs), and Akt in the presenceof NDGA. Results provided here show that IGF-IR blockade mediated byNDGA resulted in decreased proliferation, increased apoptosis, decreasedmotility, and decreased tumor growth in xenograft models. Further, theresults provided here show that NDGA is a potent inhibitor ofneuroblastoma growth and survival, and of IGF-I-stimulated signaling,events associated with tumorigenesis in neuroblastoma.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

Example 1 Breast Cancer

Materials and Methods

Materials.

NDGA and IGF-1 were gifts from Insmed Inc. (Glen Allen, Va.). IGF-IRkinase domain peptide was obtained from Upstate, USA (Charlottesville,Va.). Antibodies against the IGF-1R (C-20), HER2/neu (C-18), andphosphospecific antibodies recognizing phosphotyrosine (PY20),phosphoBAD (ser136), and pNeu (Tyr1248), and HRP-conjugatedanti-phosphotyrosine antibody (PY20HRP) were obtained from Santa CruzBiotechnology (Santa Cruz, Calif.). αIR3, a monoclonal antibody againstthe IGF-1R, was obtained from CalBiochem (San Diego, Calif.), and thephosphospecific antibody pAkt(ser473) was obtained from Cell Signaling(Beverly, Mass.). All other reagents were from Sigma (St. Louis, Mo.),except as indicated below.

IGF-1R Peptide Autophosphorylation.

1 μg of IGF-1R kinase domain peptide was incubated +/− varyingconcentrations of NDGA in 2% DMSO in 40 mM Tris, pH 7.4, 80 μM EGTA,0.25% 2-mercaptoethanol, 80 μM Na₃VO₄, 10 mM MgCl₂, and 2 mM MnCl₂ for20 min. ATP was then added at a final concentration of 20 μM.Autophosphorylation of peptide was allowed to occur for 20 min at 22° C.The reaction was stopped by the addition of SDS-reducing buffer, and thesamples were run on SDS-PAGE. Following transfer to nitrocellulosemembrane, peptide autophosphorylation was determined by western blottingemploying an antibody against phosphotyrosine diluted to 0.5 μg/ml inphosphate buffered saline (PBS) containing 3% milk. The membrane wasincubated with the antibody overnight at 4 C. Next, blots were washed 3times with PBS, then incubated with HRP conjugated sheep anti-rabbit IgG(Amersham, Piscataway, N.J.) diluted 1:50,000 in PBS with 3% milk for 90minutes at room temperature. After washing, blots were incubated withSuperSignal (Pierce, Rockford, Ill.), and exposed to film. Values weredetermined by scanning densitometry.

Preparation of Partially Purified RTKs by Wheat Germ Agglutin(WGA)-Chromotography.

IGF-1 and HER2/neu receptors were partially purified by WGAchromatography of cells overexpressing the protein of interest. Toisolate IGF-1R, CHO cells transfected with and overexpressing the humanIGF-1R (CHOIGF-1R) were plated in T-150 flasks and grown in DMEMsupplemented with 10% fetal calf serum (FCS) until 80% confluent. Cellswere harvested in the basal state and solubilized in 50 mM HEPES, 10%glycerol, 1% Triton X-100, 150 mM NaCl, 5 μM EGTA, 0.24 mg/mlaminoethyl-benzensulfonyl fluoride (AEBSF), 10 μg/ml aprotinin, 25 mMbenzamidine, 10 μg soybean trypsin inhibitor, and 5 μg/ml leuptin forone hour at 4° C. Samples were centrifuged for 60 minutes at 100,000 gand solubilized extract collected. Rodent HER2/neu receptors werecollected from MCNeuA cells grown under identical conditions andprocessed similarly. Lysates from both cell lines were loaded onto a 1ml wheat germ agglutin (WGA) column (Pharmacia, Piscataway, N.J.),washed with WGA buffer (50 mM HEPES pH 7.6, 150 mM NaCl, 0.1% TritonX-100, 1 mg/ml bacitracin, and 1 mM PMSF). Receptors were then elutedwith WGA buffer supplemented with 0.3 N N-Acetyl-D-glucosamine. WGAfractions containing RTK of interest were determined by SDS page and awestern blot that employed antibodies against either the IGF-1R orHER2/neu as appropriate.

Determination of the Effects of NDGA on the Tyrosine Kinase Activity ofPartially Purified IGF-1 R.

The effects of NDGA on the ability of the IGF-1R to phosphorylateexogenous substrates were determined by ELISA. Tyrosine kinase substratepoly Glu4:Tyr1 (PGT) was coated on Nunc-Immuno 96-well plate at 500ng/well over night at 4 C. The plate was washed and blocked withSuperblock (Pierce, Rockford, Ill.) for 30 min. WGA preparationsenriched in IGF-1R from CHO-IGFR cells were then incubated with orwithout NDGA in the presence of 10 uM ATP plus or minus 10 nM IGF-1 inKinase Buffer (50 mM, pH 7.4, 150 mM NaCl, 0.1% Triton X-100, 0.1%gelatin, 5 mM MnCl2, 8 mM MgCl2, and 1 mM PMSF). This reaction mixturewas then added to the substrate-coated wells to interact with PGT for 30min at RT. Plates were washed five times, then incubated withHRP-conjugated anti-phosphotyrosine antibody (0.3 μg/ml), diluted inSolution B (50 mM HEPES, pH 7.6, 150 mM NaCl, 0.05% Tween-20, 1 mM PMSF,2 mM vanadate and 1 mg/ml bacitracin), for two hours at 22° C. Plateswere washed and incubated with p-Tyr HRP conjugated antibody. Afterwashing, wells were incubated with 3,3′,5,5′-tetramethly benzidine (TMB)peroxidase substrate. The reaction was terminated with 1.0 M H₃PO₄.Values for receptor autophosphorylation were deter mined by measuringabsorbance at 451 nm.

Determination of the Effects of NDGA on Autophosphorylation of IsolatedHER2/neu.

Autophosphorylation of HER2/neu in WGA preparations enriched in this RTKwere determined in the absence of ligand. Samples (1-5 μl) wereincubated in the presence of varying amounts of NDGA in kinase bufferfor 15 minutes at 4° C. ATP (10 μM final concentration) was added to thereaction mixture for 45 minutes at 22° C. The autophosphorylationreaction was stopped by the addition of SDS-reducing buffer, and thesamples were run on SDS-PAGE. Following transfer to nitrocellulosemembrane, receptor autophosphorylation was determined by a western blotthat employed a phosphospecific antibody against the pNeu (Tyr1248)diluted 1:1000 in Superblock. Following an overnight incubation,membranes were washed with 3 times with tris buffered saline with 0.5%Tween 20 (TBST), then incubated with HRP conjugated sheep anti-rabbitIgG diluted 1:10,000 in Superblock for 90 minutes at room temperature.After washing, blots were incubated with SuperSignal, and exposed tofilm. Values were determined by scanning densitometry.

Determination of Ligand-Stimulated IGF-IR Signaling in Breast CancerCells.

Assays were conducted with either MCF-7, MCNeuA, or SK-Br3 breast cancercells. The MCNeuA cell line is a mammary carcinoma cell line we haverecently established from a spontaneously arising tumor in a neutransgenic female mouse (Campbell M J, Wollish W S, Lobo M, Esserman LJ: Epithelial and fibroblast cell lines derived from a spontaneousmammary carcinoma in a MMTV/neu transgenic mouse. In Vitro Cell Dev BiolAnim 38:326-333, 2002) and is thus driven by HER2/Neu overexpression.SK-Br3 human breast cancer cells express both the IGF-1R and theHER2/Neu receptor. For the initial screening, MCF-7 cells were grown in96 well plates. For dose effects of NDGA on cellular IGF-1R signaling,MCF-7, MCNeuA, or SKBR3 cells were grown in 6-well plates. For allstudies, when cells reached 80% confluence they were serum-starved for18 hr. NDGA was dissolved in DMSO and diluted with culture medium beforebeing added to cells for 1 hour at 37° C. The final concentration ofDMSO during the incubation was 0.3%. Cells were then stimulated with 3nM IGF-I for 10 minutes at 37° C. Reactions were terminated by rapidlyaspirating medium and washing cells three times with ice cold PBS. Cellswere harvested and solubilized in 50 mM HEPES, 150 mM NaCl, 1% TritonX-100, 1 mM PMSF, and 2 mM vanadate for 1 hour at 4° C. Protein wasdeter mined by BCA assay (Pierce, Rockford, Ill.)

IGF-1R autophosphorylation was determined by ELISA as describedpreviously for the IR (Youngren J F, Goldfine I D, Pratley R E:Decreased muscle insulin receptor kinase correlates with insulinresistance in normoglycemic Pima Indians. Am J Physiol 273:E276-E283,1997). Briefly, 20 μg lysate protein was added to duplicate wells in a96-well plate coated with monoclonal antibody to the IGF-IR (αIR3; 2μg/ml), and incubated 18 hours at 4° C. ELISA color development was asdescribed for the substrate phosphorylation assay.

Inhibition of IGF-1-stimulated activation of serine kinase Akt wasdetermined in the lysates prepared for the IGF-1R phosphotyrosine ELISAdescribed above. 12 μg of sample was subjected to SDS-PAGE, transferredto a nitrocellulose membrane, and phosphorylated HER2/neu quantified byblotting overnight at 4 C with a phospho-specific antibody to Akt(ser473) diluted 1:1000 in Superblock. Next, blots were washed with 3times with TBST, then incubated with HRP conjugated sheep anti-rabbitIgG diluted 1:10,000 in Superblock for 90 minutes at room temperature.Phosphorylation of the apoptotic protein BAD was determined in the samemanner by blotting with a phospho-specific antibody to BAD (ser136)(1:1,000).

Determination of NDGA Effects on Cellular HER2/neu Autophosphorylation.

HER2/neu receptor autophosphorylation was determined in serum-starvedMCNeuA or SKBR3 cells, which were collected in the basal state, due tothe ligand-independent nature of HER2/neu activation. Following aone-hour incubation in serum-free media, soluble extracts of cells wereprepared as above. HER2/neu autophosphorylation was determined by awestern blot that employed a phosphospecific antibody, pNeu(Tyr1248) asdescribed above.

Effects of NDGA on Proliferation of Breast Cancer Cells.

The inhibitory effects of NDGA on breast cancer cell growth weredetermined using a CyQUANT cell proliferation assay kit (MolecularProbes, Eugene, Oreg.). MCF-7 or MCNeuA cells were plated in 96 wellplates (5×10³ cells/well) in DMEM supplemented with 10% FCS. One platewas prepared for each harvest day. Cells were allowed to adhereovernight and were then treated with various concentrations of NDGA orDMSO as a vehicle control. Microplate cultures were harvested on days 0,1, 2, and 3 by inverting the microplate onto paper towels with gentleblotting, to remove growth medium without disrupting adherent cells.Each plate was kept at −80° C. until the end of the experiment (day 3)when all of the plates were thawed and assayed together. After thawing,200 μl of CyQUANT GR solution was added to each well and the plates wereincubated in the dark for two to five minutes. Fluorescence was measuredwith a SpectraMax Gemini XS fluorescence microplate reader (MolecularDevices, Sunnyvale, Calif.) with 480-nm excitation and 520-nm emission.Proliferation index was calculated as the percent of nucleotide contentversus control cells at day 0.

Effects of NDGA on IGF-1 Stimulated Proliferation of Breast CancerCells.

MCF-7 cells were harvested at an early passage number by washing threetimes with PBS and trypsinizing with 1 mL 0.05% trypsin. Cells wereresuspended in 5 mL defined medium (1:1 Ham's F12: DMEM 4.5 g/L glucose;1 mg/mL BSA; 10 ug/mL Transferrin; 15 mM HEPES pH 7.2; 2 mM L-glutamine;100 units/mL Penicillin G; 100 mcg/mL Streptomycin SO4; 2.5 ug/mLFungizone) containing 200 ug soybean trypsin inhibitor. Cells wereplated in 96 well collagen coated plates (Sigma, St. Louis, Mo.) at adensity of 5000 cells/well in 100 ul medium. Twenty hours later definedmedium with or without IGF-I at varying concentrations was added. Fourhours later NDGA diluted in defined medium was added. Plates wereharvested on day 3, as described above, for determination of cell numberby CyQUANT assay.

In Vivo Studies

All animal studies complied with protocols approved by of theInstitutional Animal Care and Use Committee of the University ofCalifornia, San Francisco. Our syngeneic model studies utilized theFVB/N-TgN(MMTVneu)202 mouse strain developed by Muller and colleagues(Guy C T, Webster M A, Schaller M, Parsons T J, Cardiff R D, Muller W J:Expression of the neu protooncogene in the mammary epithelium oftransgenic mice induces metastatic disease. Proc Natl Acad Sci USA89:10578-10582, 1992). This strain, denoted hereafter as neuTg,expresses the wild-type rat neu proto-oncogene (a homologue of humanHER2) under the control of the mouse mammary tumor virus (MMTV) longterminal repeat (LTR) on an FVB mouse background. The MCNeuA mammarycarcinoma cell line employed in these studies was derived from aspontaneously arising tumor in a neuTg female mouse (Campbell M J,Wollish W S, Lobo M, Esserman L J: Epithelial and fibroblast cell linesderived from a spontaneous mammary carcinoma in a MMTV/neu transgenicmouse. In Vitro Cell Dev Biol Anim 38:326-333, 2002). These cells aretumorigenic when transplanted back into neuTg mice. Female mice wereinjected subcutaneously with 10⁵ MCNeuA tumor cells on day 0. Treatmentwith NDGA began on day 9. One group of mice received 37.5 mg/kg NDGAprepared in 20% warm (37° C.) ethanol, administered intraperitonealy(i.p.) three times per week. The general preparation of NDGA intovehicle involved heating 100% ethanol and deionized distilled water to37° C. The desired amount of NDGA was then dissolved in 100% ethanol anddiluted with water drip by drip for a final solution of 20% ethanol/80%water. This solution was kept warm at 37° C. until given to mice by i.p.injection. A second group of mice received an oral dose of 100 mg/kg bygavage. NDGA was prepared for oral delivery by dissolving it incarboxymethylcellulose. Control mice received i.p. injections ofethanol/water vehicle only. Tumor growth was measured at the time ofdrug delivery on alternate treatment days with calipers and volumecalculated using the equation:

(length×width²)*(π/6).

RTK Activation in Tumors

At the end of the study, approximately 16 hrs following a final i.p.dose of NDGA, tumors were excised from the mice in order to assess theautophosphorylation state of IGF-1 and HER2/neu receptors. Tumors werehomogenized in 50 mM HEPES, pH 7.6, 150 mM NaCl, 1 mM PMSF, 2 μMleupeptin, 2 μM Pepstatin A, and 2 mM Na₃VO₄, and solubilized by theaddition of 1% Triton x-100. Soluble lysates were prepared as describedfor cell culture studies. Tyrosine phosphorylation of IGF-1R wasdetermined by ELISA as described for MCF-7 cells. Phosphorylation ofHER2/neu was determined by western blot employing the phospho-specificantibody as in the cell culture studies. The degree of phosphorylationfor each receptor was normalized to total receptor protein content inthe tumor samples as determined by western blotting with antibodiesagainst the human IGF-1R and the rodent HER2/neu.

Statistics

Statistics were calculated using MedCalc statistical software (MedCalcSoftware, Mariakerke, Belgium). Growth of breast cancer cells in cultureand in vivo was analyzed by two-way analysis of variance for treatment,time, and interaction effects with post-hoc analysis by Student'st-test. Dose effects of NDGA on receptor phosphorylation were analyzedby one-way analysis of variance with post-hoc analysis by Student'st-test. Significance was set at P<0.05.

Results Effects of NDGA on Isolated RTKs.

NDGA (FIG. 1) has previously been shown to directly inhibitligand-stimulated PDGFR autophosphorylation (Domin J, Higgins T,Rozengurt E: Preferential inhibition of platelet-derived growthfactor-stimulated DNA synthesis and protein tyrosine phosphorylation bynordihydroguaiaretic acid. J Biol Chem 269:8260-8267, 1994) and ahomologous compound is an inhibitor of the IGF-1R (Blum G, Gazit A,Levitzki A: Substrate competitive inhibitors of IGF-1 receptor kinase.Biochemistry 39:15705-15712, 2000; Blum G, Gazit A, Levitzki A:Development of new insulin-like growth factor-1 receptor kinaseinhibitors using catechol mimics. J Biol Chem 278:40442-40454, 2003).

The present invention shows that NDGA has a direct effect on both IGF-1Rand HER2/neu receptors. More specifically, the present invention showsthe ability of NDGA to inhibit autophosphorylation and/or substratetyrosine kinase activity of both these receptors in vitro. The abilityof NDGA to directly inhibit the kinase domain of the receptor was shownusing a synthetic peptide consisting of the 379 terminal amino acids ofthe IGF-1R beta subunit. This peptide, which displays intrinsicautophosphorylation and substrate tyrosine kinase activity, wasincubated with increasing concentrations of NDGA prior to the additionof 20 μM ATP. Under these conditions, NDGA inhibited activation of theIGF-1R kinase domain at a concentration at 1 μM and the results areshown in the Western blot image of FIG. 8.

The effects of NDGA against the intact IGF-1 and HER2/neu receptors wereshown using wheat germ affinity chromatography to obtain fractionsenriched in either the human IGF-1R or the rodent HER2/neu receptorextracted from cells overexpressing these proteins. In IGF-1Rpreparations, we determined the ability of NDGA to inhibit IGF-1stimulated phosphorylation of the synthetic tyrosine kinase substrate,poly Glu4:Tyr1. Incubation of IGF-1R preparations with NDGA for 20minutes prior to the addition of 10 nM IGF-1 produced dramaticreductions in tyrosine phosphorylation of the substrate (FIG. 3). Thiseffect was observed at concentrations of NDGA as low as 0.3 μM.

Because HER2/neu is intrinsically active in cells in the absence ofligand binding, basal autophosphorylation is observed in isolatedreceptor preparations (FIG. 4). Incubation of HER2/neu preparations with10 μM ATP produced a further increase in ligand-independent HER2/neuautophosphorylation. However, preincubation with NDGA for 20 minutesprior to the addition of ATP abolished this increase in HER2/neuautophosphorylation (FIG. 4).

Effects of NDGA on IGF-1R and HER2/neu Signaling in Cells.

Treatment of MCF-7 cells with increasing concentrations of NDGA for onehour prior to the addition of 3 nM IGF-1 produced a dose-dependentdecrease in IGF-1R autophosphorylation with an IC₅₀ of 31±12 μM (FIG.5A). Similar effects of NDGA on IGF-1R autophosphorylation were observedin SK-Br3 breast cancer cells (data not shown). In order to determinethe impact of NDGA on downstream signaling of the IGF-1R, we studied theAkt/BAD pathway that regulates cellular apoptosis. Incubation of MCF-7cells with 1 nM IGF-1 dramatically increased phosphorylation of Akt,and, to a lesser extent, BAD. These effects were reduced in adose-dependent manner by concentrations of NDGA similar to those thatinhibited IGF-1R autophosphorylation (FIG. 5B). These data demonstratethat NDGA treatment results in transition into a pro-apoptotic state forMCF-7 cells.

Because MCF-7 cells do not express HER2/neu, we employed MCNeuA cells, abreast cancer cell line derived from transgenic mice overexpressing therodent form of this receptor. Exposure of these cells to increasingconcentrations of NDGA produced a dose-dependent inhibition of HER2/neuautophosphorylation. Ligand-independent tyrosine phosphorylation ofHER2/neu was inhibited by NDGA with an IC₅₀ of 15±4 (FIG. 6). Similarresults were observed in SK-Br3 cells, which express relatively largeamounts of the human HER2/neu receptor (data not shown).

Effects of NDGA on Growth of Breast Cancer Cells in Culture:

Given the ability of NDGA to inhibit the function of the IGF-1R and theHER2/neu receptor, we tested its effect on the proliferation of MCF-7and MCNeuA breast cancer cells under normal culture conditions. WhenMCF-7 cells were grown in 10% fetal calf serum and then incubated withvarying concentrations of NDGA for up to 3 days, the rate ofproliferation was significantly reduced by NDGA concentrations as low as15 μM (FIG. 7A). At 60 μM, cell number was dramatically reduced within24 hours. As numerous growth factors are present in serum, we examinedthe ability of NDGA to specifically inhibit growth of these cellsmediated by IGF-1 alone. In MCF-7 cells grown in media supplemented onlywith 10 nM IGF-1, NDGA inhibited proliferation with an IC₅₀ ofapproximately 10 μM (FIG. 7B). In the presence of 10% fetal calf serum,NDGA also inhibited growth of MCNeuA cells, for which growth is largelydue to the ligand-independent activity of the overexpressed HER2/neureceptor. and SK-Br3 cells, which express relatively high levels of bothIGF-1 and HER2/neu receptors with potencies similar to that observed forMCF-7 cells (data not shown).

Effects of NDGA on IGF-1R and HER2/neu Receptor Activation of BreastTumors in Mice:

In order to assess the ability of NDGA to inhibit activation of theIGF-1R and HER2/neu receptor in vivo, we employed a syngeneic mousemodel of breast cancer featuring a cell line that expresses both RTKs.In these studies, MCNeuA breast cancer cells were injectedsubcutaneously into female neuTg mice, the strain from which this cellline was originally obtained. NDGA was administered three times a weekeither as an i.p. dose of 37.5 mg/kg or as an oral dose of 100 mg/kg,prepared in carboxymethylcellulose. Administration of NDGA began ninedays after implantation of tumor cells. Twenty nine dayspost-implantation, and 16 hours following the final administration ofNDGA tumors were excised from intraperitonealy-treated and control miceat the end of the study and analyzed. We observed thatautophosphorylation of both receptors was reduced in tumors fromNDGA-treated mice compared to vehicle-treated controls (FIG. 8). NDGAtreatment had no effect on the total cellular content of either theIGF-1 or HER2/neu receptor.

FIG. 8 shows that NDGA Directly Inhibits Autophosphorylation of IGF-1RKinase Domain. A peptide corresponding to the kinase domain of IGF-1Rwas incubated with varying concentrations of NDGA prior to the additionof ATP. Inhibition of peptide tyrosine phosphorylation by NDGA wasdetermined by western blots that employed an anti-phosphotyrosineantibody. A representative blot is shown.

Inhibition of signaling by both RTKs through treatment with NDGA in vivowas associated with a reduced tumor growth rate. There were nodifferences in tumor growth rates between animals receiving oral or i.p.administration of NDGA. Data combined from both treatment groupsdemonstrated that NDGA significantly reduced tumor growth from 21 dayspost-implantation through the remainder of the study (FIG. 9).

FIG. 9 shows that NDGA Directly Inhibits Tyrosine Kinase Activity ofIsolated IGF-1R. IGF-1R were partially purified by affinitychromatography from cells overexpressing human IGF-1R. The IGF-1R wasincubated with varying concentrations of NDGA prior to the addition ofIGF-1. ATP-induced tyrosine phosphorylation of the immobilizedsubstrate, poly Glu4:Tyr1 was determined by ELISA employing ananti-phosphotyrosine antibody to readout. IGF-1 stimulation of substratephosphorylation was calculated as the O.D. value in the absence of addedIGF-1 subtracted from the O.D. value in each experimental condition.Values represent mean±SEM of 3 experiments.

FIG. 10 shows that NDGA Directly Inhibits Autophosphorylation ofIsolated HER2/neu. HER2/neu receptors were partially purified byaffinity chromatography from cells overexpressing mouse HER2/neu.Incubation of HER2/neu receptors with NDGA prior to the addition of ATPreduced autophosphorylation as determined by western blot employing aphosphospecific antibody, pNeu(Tyr1248). A representative blot of 3experiments is shown.

FIG. 11 shows that NDGA Inhibits the IGF-1R Signaling Pathway in MCF-7Cells. MCF-7 breast cancer cells were incubated with varyingconcentrations of NDGA for 1 hr. Soluble extracts were then collected inthe basal state or following a 10 minute incubation with 3 nM IGF-1. (A)Tyrosine phosphorylation of the IGF-1R was then determined by specificELISA (▪ basal state; 3 nM IGF-1). Values represent mean±SEM of 3experiments, normalized to cells treated with vehicle alone. *Valuessignificantly reduced vs. vehicle treated controls. (B) Serinephosphorylation of PKB and BAD determined by western blot.Representative blots of 3 experiments are shown.

FIG. 12 shows that NDGA Inhibits HER2/neu Autophosphorylation in MCNeuACells. MCNeuA breast cancer cells were incubated with varyingconcentrations of NDGA for 1 hr. Tyrosine phosphorylation of HER2/neureceptors in soluble extracts was determined by western blot employing aphosphospecific antibody. A representative blot of four experiments isshown.

FIG. 13 shows that NDGA Inhibits Serum and IGF-1 StimulatedProliferation of MCF-7 Breast Cancer Cell Lines. (A) MCF-7 cells weregrown in media supplemented with 10% fetal calf serum. Beginning on day0, cells were incubated with varying concentrations of NDGA. Cell numberwas estimated by determination of nucleotide content (CyQUANT assay) ondays 0, 1, 2, and 3. Proliferation index was calculated as the percentdifference in cell number vs. day 0. (B) MCF-7 cells were initiallyplated in basal media. Growth continued in basal media or mediasupplemented with 10 nM IGF-1. 24 hours after plating (day 0), cellswere incubated with varying concentrations of NDGA or DMSO alone. Plateswere harvested on day 3 for CyQuant assay. Values shown are O.D. valuesreflecting total nucleic acid content per well. All values representmean±SEM of 3 experiments. *Proliferation significantly reduced vs.vehicle treated controls. *Cell number significantly reduced from day 0.

FIG. 14 shows that Chronic NDGA Administration Inhibits RTK Activationin Tumors In Vivo. Following 21 days of treatment, and 16 hrs after thefinal intraperitoneal administration of NDGA, MCNeuA tumors were excisedand soluble protein extracts prepared. Tyrosine phosphorylation ofIGF-1R (black bars) was deter mined by ELISA. Phosphorylation ofHER2/neu (gray bars) was determined by western blot. Tumor content ofboth RTKs was not different across treatment groups as determined bywestern blot. Values represent mean phosphorylation level normalized tocontrol values±SEM for 4-5 animals per group. *Receptor tyrosinephosphorylation significantly reduced vs. vehicle treated controls.

FIG. 15 shows that Growth Inhibition of MCNeuA Cells in vivo By NDGAAdministered Orally and by Intraperitoneal Injection. MCNeuA cells wereinjected into NeuTG mice on day 0. Treatment with NDGA began on Day 9,with NDGA administered 3× week, either orally in carboxymethylcellulose(100 mg/kg) (▴) or injected intraperitoneally (37.5 mg/kg) (▪). Valuesrepresent mean tumor volume±SEM for 4-5 animals per group. *Tumor volumesignificantly reduced for combined treatment groups vs. vehicle treatedcontrols.

Example 2 Neuroblastoma Materials and Methods

Cell culture and reagents. Human SH-SY5Y, SHEP, and Kelly neuroblastomacells were cultured in Dulbecco's modified Eagle medium (DMEM) with 10%calf serum and maintained in a humidified incubator with 10% CO₂ at 37°C. NDGA from Insmed Corporation (Richmond, Va.) was dissolvedimmediately before each experiment in DMSO to make a 1000× solution,which was then added to the cell culture medium. IGF-I was purchasedfrom GroPep (Adelaide, S A, Australia). Anti-IGF-IR antibody (αIR-3) waspurchased from Calbiochem (San Diego, Calif.). Anti-phosphotyrosineantibody was purchased from Santa Cruz Biotechnology (Santa Cruz,Calif.). Anti-Akt, anti-phospho-Akt, anti-Erk1/2, anti-phospho-Erk1/2,and anti-cleaved caspase-3 antibodies were purchased from Cell SignalingTechnologies (Beverly, Mass.). Horseradish peroxidase conjugated goatanti-rabbit IgG was purchased from Zymed Laboraties (South SanFrancisco, Calif.). CyQuant was purchased from Molecular Probes (Eugene,Oreg.). Propidium iodide was purchased from Sigma (St. Louis, Mo.).

IGF-IR phosphorylation ELISA. SH-SY5Y and SHEP cells were grown to 80%confluence in DMEM/10% calf serum, then serum-starved for 4 h. Cultureswere then treated with DMSO or 60 uM NDGA and incubated for 1 h. Somecultures were then treated with 1 nM IGF-I for 10 min. The medium wasremoved, cultures rinsed 3× in cold PBS, and lysis buffer (120 mM HEPES,300 mM NaCl 2 mM sodium orthovanadate, and 1 mMphenylmethylsufonylfluoride (PMSF)) was added. Cultures were rocked inlysis buffer at 4° C. for 1 h. 96-well plates were coated with αIR-3antibody in 50 mM NaHCO₃, pH 9.0, for 2 h at RT. Plates were rinsed 3xin tris-buffered saline+0.1% Tween (TBST), then blocked with SuperBlock(Pierce, Rockford, Ill.) for 30 min at RT. Each well of the ELISA platereceived 30 μg of lysate protein from the cell cultures, followed by 24h incubation at 4° C. Plates were rinsed 5× with TBST, andHRP-conjugated anti-phosphotyrosine antibody was added (1:2000, dilutedin 120 mM HEPES, 300 mM NaCl, 2 mM sodium orthovanadate, and 1 mM PMSF,1% bovine serum albumin, 1 mg/ml bacitracin, and 0.5% Tween-20) for 2 hat RT. Plates were again rinsed 5× in TBST, and TMB (Pierce) was addeduntil blue color was sufficiently developed. Absorbance at 451 nm wasquantified. Each lysate was run in triplicate, and the experiment wasrepeated 3 times.

CyQUANT assay for cell growth. Cells were plated on four 96-well tissueculture plates, in DMEM/10% calf serum at a density of 8000 cells/welland incubated for 24 h. In one set of experiments, the medium wasswitched to serum free DMEM supplemented with 1% bovine serum albumin(to provide osmotic support). IGF-I (10 nM) was added to some samples ona daily basis for up to 3 days. In a second set of experiments, thecells continued to be cultured in DMEM/10% calf serum for the durationof the experiment, with no additional IGF-I. For all experiments, DMSOor different concentrations of NDGA were added to the cultures at 0 h(the day after plating). The media from one plate was immediatelyremoved, and the plate was frozen at −80° C. This plate served as thebaseline for the experiment. Single plates were frozen at 24, 48, and 72h following addition of drugs. DNA content of each well was quantifiedby staining with CyQUANT according to the manufacturer's instructionsand measuring CyQUANT absorbance with a fluorimeter. Each condition wasrun in triplicate, and the experiment was repeated three times.

Detection of phospho-Akt and phospho-Erk. Cells were grown to 80%confluence, serum starved for 4 h, and treated with DMSO or differentconcentrations of NDGA for 1 h. Then, some cultures were treated with 10nM IGF-I for 15 min. Cultures were immediately placed on ice, the mediumwas removed, and the cells were lysed in modified RIPA buffer (20 mMTris, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 0.1% sodiumdodecyl sulfate, and 1% deoxycholate). Fifty μg of protein from eachsample were separated via SDS-PAGE and transferred to nitrocellulose.Phospho- and total-Akt and -Erk were detected by immunoblotting.

Propidium iodide staining of apoptotic cells. SH-SY5Y cells werecultured in DMEM with 10% calf serum. Cultures were treated with DMSO orNDGA for 24 h. The supernatant was collected to save detached cells.Attached cells were removed from the plate via trypsinization, andpelleted by centrifugation in combination with the cells in thesupernatant. The cell pellet was fixed by drop-wise addition of cold 70%ethanol while vortexing gently, and stored at 4° C. The pellet waswashed twice and resuspended in PBS and stained with 1 μg/ml propidiumiodide. Propidium iodide fluorescence was measured in 30,000 cells persample using a Becton-Dickinson (Franklin Lakes, N.J.) Facscalibur flowcytometer. The percentage of cells in each stage of the cell cycle, aswell as the percentage of cells that were apoptotic (sub-G0) wasdetermined by analyzing the data with ModFit software. The experimentwas repeated three times.

Detection of Caspase-3 cleavage. Neuroblastoma cells were grown to 80%confluence and treated with DMSO or NDGA for 6 h. Alternatively, cellswere serum starved for 4 h and cultured with or without 60 μM NDGA andwith or without 10 nM IGF-I for 3 h. Lysates were collected as describedabove, and the 14/17 kD cleavage fragments of caspase-3 were detectedvia SDS-PAGE followed by immunoblotting with anti-cleaved caspase-3antibody.

Measurement of cell motility. Neuroblastoma cells were plated on goldparticle-coated coverslips (prepared as described in (30)) in serum-freemedia at a density of 25,000 cells per coverslip. The cells wereincubated for 2 h to allow adhesion to the coverslip. Then, wells weretreated with DMSO or 30 μM NDGA for 1 h. 1 nM IGF-I was then added tosome wells. Incubation continued for 6 h, followed by fixation with 3.5%glutaraldehyde. Coverslips were mounted on glass slides, then viewed ona Lietz Orthoplan inverted microscope attached to a Sony videoprocessor.Digital images of the tracks etched into the gold by the cells from 3separately treated coverslips per condition were collected at 200×magnification using Adobe Photoshop software. For each condition, theareas of 120 tracks made by individual cells were measured with NIHImage 1.61 software.

Treatment of xenografted nude mice with NDGA. Nude mice xenografted withhuman Kelly neuroblastoma cells were treated with NDGA to determine ifNDGA can affect tumor growth in vivo. Briefly, 7×10⁶ Kelly humanneuroblastoma cells were resuspended in a 1:1 mixture of PBS andMatrigel (BD Clontech Inc.) and 100 μl of the mixture was injectedsubcutaneously into the flanks of 6-12 week-old BALB/c nude mice. After1 cm tumors were established (˜10-14 days post implantation), animalswere injected subcutaneously with either vehicle (DMSO) or NDGA, (50mg/kg, suspended in DMSO) daily for 10 days. Tumors were then harvested,weighed and measured. The formula width²×length/2 was used to calculatetumor volumes.

Results

IGF-I stimulated IGF-IR phosphorylation in neuroblastoma cells isinhibited by NDGA. NDGA was previously shown to inhibit theautophosphorylation of the IGF-IR in partially purified preparations ofthe receptor, and in breast cancer cell lines. To test the effects ofNDGA on IGF-IR activation in neuroblastoma cells, serum-starved SHEP andSH-SY5Y neuroblastoma cells were treated with either DMSO (vehiclecontrol) or 60 M NDGA for 1 h (FIGS. 16A and 16B). The cultures werethen treated for 10 min with or without 1 nM IGF-I. Protein lysates werecollected and the degree of IGF-IR phosphorylation in the lysate sampleswas quantified using ELISA. IGF-I induced an increase in IGF-IR tyrosinephosphorylation in both cell lines. This IGF-I stimulated receptorphosphorylation was inhibited by NDGA. SH-SY5Y (A) cells showed a higherresponse to IGF-I than SHEP cells (B), consistent with the increasedbasal levels of IGF-IR found in SH-SY5Y cells (Kim B. van Golen C M andFeldman E L. Insulin-like growth factor-I signaling in humanneuroblastoma cells. Oncogene 2004;23:130-141).

FIGS. 16A and 16B show that NDGA inhibits activation of the IGF-IR byIGF-I. SH-SY5Y (A.) and SHEP (B.) neuroblastoma cells were serum starvedand treated with DMSO or 60 μM NDGA for 1 h. Then, 1 nM IGF-I was addedto half the cultures for 10 min. IGF-IR phosphorylation was detected byan ELISA (Materials and Methods). Results are means +/− SEM formeasurements collected in all experiments expressed as a percentage ofIGF-IR phosphorylation in unstimulated DMSO-treated cells. Theexperiment was repeated three times, with each condition run intriplicate within each experiment. *p<0.01 vs. DMSO+IGF-I.

NDGA inhibits neuroblastoma proliferation The amount of cellular DNApresent in neuroblastoma cultures was measured to quantify the cellproliferation or cell death that occurred during a three day treatmentwith NDGA. SH-SY5Y and Kelly neuroblastoma cells were cultured on four96-well plates in serum-free media supplemented with 10 nM IGF-I for upto 72 h. At 0 h, the cultures were treated with 15-120 μM NDGA, or DMSOas control. The cell content of each plate was measured by staining fortotal DNA, using CyQUANT dye. In both SH-SY5Y and Kelly cells, treatmentwith NDGA inhibited proliferation, and caused cell death at higher doses(FIGS. 17A and 17B).

FIGS. 17A and 17B show that NDGA inhibits neuroblastoma growth inserum-free medium supplemented with 10 nM IGF-I. SH-SY5Y (A.) and Kelly(B.) neuroblastoma cells were cultured in serum-free medium with 10 nMIGF-I and treated with DMSO or NDGA (15-120 μM). DNA content wasmeasured at 0, 24, 48, and 72 h using CyQUANT staining (Materials andMethods). Means +/− SEM from three separate experiments are expressed asa percentage of absorbance at 0 h. *p<0.05 vs. DMSO-treated control atthe same time point.

To determine if NDGA would still have an inhibitory effect onneuroblastoma growth in serum, where other factors could contribute toneuroblastoma mitogenesis and survival in addition to IGFs, theexperiment was repeated using SHEP and SH-SY5Y cells cultured in mediumcontaining 10% serum (FIGS. 18A and 18B). NDGA inhibited theproliferation of SH-SY5Y and SHEP cells cultured in serum up to 72 h.Higher doses of NDGA caused cell death, as the amount of DNA in thesecultures is less than the amount at 0 h. These results demonstrate thatNDGA inhibits the growth and survival of neuroblastoma cells supportedby either serum or IGF-I.

FIGS. 18A and 18B show that NDGA inhibits neuroblastoma growth in serum.SH-SY5Y (A.) and SHEP (B.) neuroblastoma cells were cultured in serumand treated with DMSO or NDGA (30-120 μM). Samples were collected andprocessed for CyQUANT absorbance as in FIG. 2. Each bar represents themean +/− SEM of three separate experiments, and each condition was runin triplicate. *p<0.05 vs. DMSO-treated control at the same time point.

NDGA prevents IGF-I activation of the MAPK pathway. Neuroblastomamitogenesis is regulated by IGFs via the activation of the MAPKsignaling pathway, leading to the phosphorylation and activation of ERK1 and 2. The effects of NDGA treatment on IGF-stimulated ERKphosphorylation were investigated in SI-WP and SH-SY5Y neuroblastomacells. Serum starved cells were treated for 1 h with DMSO or increasingconcentrations of NDGA, and then stimulated with 10 nM IGF-I for 15 min.Lysates were collected and proteins separated by SDS-PAGE as describedin Materials and Methods. ERK phosphorylation was assessed byimmunoblotting with anti-phospho ERK1/2 antibody. ERK phosphorylationwas increased by IGF-I in SH-SY5Y cells (FIG. 19A). NDGA causes adose-dependent inhibitory effect on IGF-stimulated phosphorylation ofERK. The total ERK content of each lane is shown for comparison. Similarresults were obtained in SHEP cells.

Akt phosphorylation is inhibited by NDGA. IGFs also signal through thePI-3K pathway in neuroblastoma cells, leading to the activation of Akt.The effect of NDGA on IGF-stimulated Akt activation was assessed inserum-starved SH-SY5Y and SHEP cells, via SDS-PAGE and Westernimmunoblotting. Similar to the effects on ERK phosphorylation, NDGAcaused a dose-dependent inhibition of IGF-stimulated Akt phosphorylationin SH-SY5Y and SHEP cells (FIG. 19B). Total Akt content of each lane isshown for control.

FIGS. 19A and 19B show that IGF-I-stimulated ERK and Akt activation areblocked by NDGA. SH-SY5Y cells were serum starved and treated with DMSOor 3, 30, or 60 μM NDGA for 1 h, then given 10 nM IGF-I for 15 min.Lysates were collected and ERK (A.) or Akt (B.) phosphorylation wasdetected via Western blot analysis. A. Upper panel shows ERKphosphorylation. Lower panel is total ERK to show equal loading oflanes. B. Upper panel shows phospho-Akt, while lower panel shows totalAkt as a loading control. Representatives of three separate experimentsare shown.

Caspase-3 is activated by NDGA. Akt activation supports neuroblastomasurvival by suppressing apoptosis, in part by preventing the catalyticactivation of caspase-3. Disruption of Akt signaling increasesactivation of caspase-3 driving neuroblastoma cells into apoptosis.

To determine if NDGA causes caspase-3 activation, SH-SY5Y neuroblastomacells were cultured in serum and treated with NDGA for 6 h. Caspase-3activation was assessed by SDS-PAGE and immunoblotting with anti-cleavedcaspase-3 antibody, which detects the small cleavage fragments ofcaspase-3 that are released upon its activation. GAPDH expression wasdetected for loading control. NDGA causes dose-dependent caspase-3activation (FIG. 20A). To determine if exogenous IGF-I was able toprevent this activation, SH-SY5Y cells were cultured in serum-free mediacontaining 10 nM IGF-I and simultaneously treated with NDGA or DMSO as acontrol. Caspase-3 activation was still detectable when NDGA-treatedcells were given IGF-I (10 nM) (FIG. 20B). Caspase-3 activation was notdetected in serum-starved cells cultured in the absence of IGF-I, whileNDGA treated SH-SY5Y cells cultured in the absence of IGF-I, whichsecrete their own IGF-II, showed strong caspase activation. This showsthat NDGA is capable of both pushing the cells into an apoptotic stateand suppressing IGF-mediated rescue.

NDGA causes neuroblastoma cells to undergo apoptosis. SH-SY5Y cellscultured in serum were treated with DMSO or NDGA (30-120 μM). After 24h, the cells were harvested and subjected to flow cytometric cell cycleanalysis as described in Materials and Methods. NDGA causes a dramatic,dose-dependent increase in the percentage of sub-G₀ cells, the fractionof cells undergoing apoptosis (FIG. 20C).

FIGS. 20A, 20B and 20C show that NDGA causes caspase activation andapoptosis in neuroblastoma cells. A. SH-SY5Y cells grown in serumcontaining-medium were treated with DMSO or 0.3-60 μM NDGA for 12 h.Activated caspase-3 fragments were detected using western blot analysis.Upper panel shows the 14/17 kD cleavage fragments of caspase-3, whilethe lower panel shows GAPDH expression as a loading control. Arepresentative of three separate experiments is shown. B. Serum-starvedSH-SY5Y cells were treated with DMSO or 60 μM NDGA for 12 h. Somecultures included 10 nM IGF-I for the entire treatment period. Lysateswere collected and caspase-3 cleavage fragments were detected as above.C. SH-SY5Y cells were treated with DMSO or NDGA (30-120 μM) for 24 h,fixed, stained with propidium idodide, and subjected to flow cytometricanalysis of cell cycle. Bars represent the mean +/− SEM percentage ofcells in the sub-G₀ apoptotic phase from five separate experiments.*p<0.05 vs. DMSO.

IGF-stimulated cell motility is inhibited by NDGA. IGFs increase themotility of neuroblastoma cells, in part through PI-3K signaling. Theability of NDGA to impact neuroblastoma motility was assessed bymeasuring the motility of serum starved SHEP and SH-SY5Y cells treatedwith or without 1 nM IGF-I, over a 6 h period. Motility was quantifiedby plating the cells on coverslips coated with fine particles, and thenmeasuring the areas cleared of particles by the cells after they movedduring the 6 h incubation. IGF-I increased the motility of SH-SY5Y andSHEP cells, and 30 μM NDGA strongly suppressed this increase in motility(FIG. 21A).

NDGA inhibits tumor growth in a xenograft model of neuroblastoma. Todetermine if the anti-tumorigenic effects of NDGA would be observed invivo, nude mice with established Kelly cell xenografts were treated withNDGA (50 mg/kg i.p. daily) or vehicle (n=4 per treatment group). After10 d of treatment, all mice were sacrificed because the tumors in thevehicle treated mice had grown so large that our institutional animalcare rules required the animals be sacrificed. NDGA, however, hadinhibited tumor growth by 50% (FIG. 21B).

FIGS. 21A, 21B and 21C show that NDGA inhibits IGF-I stimulated motilityand in vivo neuroblastoma tumor growth. A. SH-SY5Y and SHEP cells wereplated on gold particle-coated coverslips in serum-free conditions.After adhering, the cells were treated with DMSO or 30 μM NDGA for 1 h.Half the cultures were then treated with 1 nM IGF-I, and incubationcontinued for 6 h. The track areas of cells that were etched into thegold particle coating were measured using NIH Image software. Each barrepresents the mean +/− SEM of 120 individual track areas collected fromthree separate experiments. *p<0.001 vs. DMSO+1 nM IGF-I. B. Kellyneuroblastoma cells were implanted subcutaneously in nude mice asdescribed in Materials and Methods. When palpable tumors formed (day12), mice were treated with daily i.p. injections of DMSO (vehicle,solid line) or 50 mg/kg NDGA (dashed line). On day 22, the animals weresacrificed and tumor volume was measured with calipers. N=4 animals ineach treatment group.

Discussion

The IGF signaling system has become a target of increasing interest incancer therapy research. A variety of approaches to disrupting thesystem have been proposed and investigated, including use ofanti-receptor antibodies, anti-sense nucleotides, and ligand mimickingcompounds (Foulstone E, Prince S, Zaccheo O, et al. Insulin-like growthfactor ligands, receptors, and binding proteins in cancer. J Pathololgy2005;205:145-153). Decreasing IGF availability to tumors is also beingconsidered. A fusion protein of the IGF binding proteins 3 and 6 isreported to sequester autocrine IGF-II and decrease rhabdomyosarcomathymidine uptake (Dake B L, Boes M, Bach L A and Bar R S. Effect of aninsulin-like growth factor binding protein fusion protein on thymidineincorporation in neuroblastoma and rhabdomyosarcoma cell lines.Endocrinology 2004;145:3369-3374). Small molecular inhibitors of theIGF-I receptor are another approach that is attracting much attention. Arelated pair of highly specific and potent inhibitors of the IGF-IR,NVP-ADW742 and NVP-AEW541, inhibit the growth of a wide variety oftumors in vitro, as well as fibrosarcoma growth in vivo (Mitsiades C S,Mitsiades N S, McMullan C J, et al. Inhibition of the insulin-likegrowth factor receptor-1 tryosine kinase activity as a therapeuticstrategy for multiple myeloma, other hematologic malignancies, and solidtumors. Cancer Cell 2004;5:221-230; Garcia Echeverria C, Pearson M A,Marti A, et al. In vivo antitumor activity of NVP-AEW541-A novel,potent, and selective inhibitor of the IGF-IR kinase. Cancer Cell2004;5:231-239). Still, relatively few agents have been identified thathave affects against the IGF-IR. Considering the promising pre-clinicalresults of anti-IGF treatment in numerous malignancies.

NDGA is a naturally occurring compound that has been extensively studiedfor its anti-lipoxygenase activity. It will inhibit the tyrosinephosphorylation of partially purified IGF-I and her2/neu receptors, aswell as these same receptors endogenously expressed in breast cancercells. While it is quite potent at inhibiting these receptors, NDGA doesnot show high selectivity for a single receptor, in contrast toNVP-ADW742 and NVP-AEW541. NDGA inhibits the activation of the PDGFreceptor and PDGF-stimulated DNA synthesis (Domin J, Higgins T, andRozengurt E. Preferential inhibition of platelet-derived growthfactor-stimulated DNA synthesis and protein tyrosine phosphorylation bynordihydroguaiaretic acid. J Biol Chem 1994:269:8260-8267). Seufferlein,et al., found no affect of NDGA on EGF receptor phosphorylation.suggesting it has some selectivity (Seufferlein T, Seckl M J, Schwarz E,et al. Mechanisms of nordihydroguaiaretic acid-induced growth inhibitionand apoptosis in human cancer cells. Brit J Cancer 2002:86:1188-1196).

Due to NDGA's effects on the structurally similar insulin receptor, adiabetic phenotype is one logical toxicity to expect. Paradoxically,NDGA has an anti-diabetic effect on rats, decreasing serum glucose andtriglycerides without affecting insulin levels. NDGA was previouslyconsidered for treatment of diabetes because of its inhibition ofprostaglandin synthesis. Thus. NDGA's inhibition of insulin receptorsmay not result in a diabetes-like toxicity because of its concomitanteffects on prostaglandin synthesis. NDGA analogs are being developed inan attempt to achieve better specificity, and some have been tested forefficacy against lung cancer (Moody T W, Leyton J, Martinez A, Hong S,Malkinson A and Mulshine J L. Lipoxygenase inhibitors prevent lungcarcinogenesis and inhibit non-small cell lung cancer growth. Exp LungRes 1998:24:617-628). Further characterization of these analogs may leadto the discovery of agents more specific for individual receptortyrosine kinases.

NDGA has already been tested as a potential anti-cancer agent in severalin vitro and in vivo studies, often with an underlying hypothesis thatsuppressing prostaglandin synthesis will suppress tumor growth withoutdirectly evaluating this mechanism of action. NDGA is effective in vitroagainst numerous tumor cell types, where it induces apoptosis andsuppresses mitogenesis (Seufferlein T, Seckl M J, Schwarz E, et al.Mechanisms of nordihydroguaiaretic acid-induced growth inhibition andapoptosis in human cancer cells. Brit J Cancer 2002;86:1188-1196; Tong W-G, Ding X -Z, Witt R C and Adrian T E. Lipoxygenase inhibitorsattenuate growth of human pancreatic cancer xenografts and induceapoptosis through the mitochondrial pathway. Mol Cancer Therap2002;1:929-935; Hoferova Z, Fedorocko P, Hofer M, Hofmanova J, Kozubik Aand Eliasova V. Lipoxygenase inhibitors suppress proliferation of G5:113fibrosarcoma cells in vitro but they have no anticancer activity invivo. Neoplasm 2003;50:102-109; Vondracek J, Stika J V, Soucek K, et al.Inhibitors of arachidonic acid metabolism potentiate tumor necrosisfactor-alpha-induced apoptosis in HL-60 cells. Eur J Pharmacol2001;424:1-11). Cancers that are highly responsive to IGF, includinglung (Moody T W, Leyton J, Martinez A, Hong S, Malkinson A and MulshineJ L. Lipoxygenase inhibitors prevent lung carcinogenesis and inhibitnon-small cell lung, cancer growth. Exp Lung Res 1998;24:617-628) andbreast, respond to NDGA treatment in vivo.

Considering its potency in inhibiting IGF-IR phosphorylation, NDGA couldbe effective in suppressing the growth of neuroblastoma tumors, whichare highly responsive to IGFs. Both paracrine and autocrine IGFsstimulate neuroblastoma mitogenesis. Neuroblastoma cell lines thatsecrete IGF-II are capable of serum-independent growth. Additionally,cell lines that express high levels of the IGF-IR are more aggressivelytumorigenic. IGFs strongly activate the MAPK signaling pathway, whichculminates in phosphorylation of ERK 1 and 2. Thus, interrupting IGFsignaling at the level of the receptor can be used to prevent the growthof neuroblastoma tumors.

The present invention shows that NDGA at low doses (15-30 μM) completelyblocks neuroblastoma growth over a period of several days in vitro, bothin serum and serum-free conditions where added and autocrine IGFssupport neuroblastoma growth. The growth of Kelly neuroblastoma tumorxenografts in nude mice is also suppressed by NDGA. A single daily doseof an NDGA formulation of the invention, was very well tolerated by themice, and inhibited tumor growth by 50%. NDGA prevents IGF-I-mediatedactivation of both the IGF-IR and ERK 1 and 2 in neuroblastoma cells atthe same doses that inhibit growth in vitro. NDGA was similarlyeffective at inhibiting IGF-IR signaling and the growth of breast cancercells in vitro and in xenografts. The effects of NDGA on other moleculartargets notwithstanding, the ability of NDGA to inhibit mitogenesis inthese experiments is likely attributable at least in part to blockingIGF-IR activation.

IGFs are also potent stimulators of neuroblastoma survival, causingstrong activation of Akt and Bcl-2 while suppressing caspase-3activation. Results provided here show that NDGA causes neuroblastomadeath as the dose is increased and is strongly apoptotic, causingcaspase-3 activation and a large increase in sub-G₀ cells. IGF-Inormally can prevent caspase activation in neuroblastoma cells, butIGF-I activation of Akt was inhibited in cells treated with NDGA.Additionally, IGF-I was only partially able to mitigate NDGA-inducedcaspase-3 activation. NDGA appears to prevent the survival-promotingeffects of IGFs by interrupting signaling pathways. Similar results areseen in breast cancer cell lines treated with NDGA, where Akt activationis suppressed and BAD activation is increased. NDGA and otherlipoxygenase inhibitors cause caspase-3 activation in pancreatic cancercells; thus, a lipoxygenase-related mechanism is also possible inneuroblastoma.

IGF-I stimulates neuroblastoma cells to undergo organized actinpolymerization and lamellipodium extension, resulting in increased cellmotility. Increased cell motility, along with the ability to digestextracellular matrix, affords cancer cells greater ability to invadetissues and blood vessels, leading to metastasis and diffuse tissuedissemination. This is of particular concern with neuroblastoma, wheretumor invasion of bone, a site of high IGF production, is associatedwith poor response to therapy. NDGA effectively inhibits IGF-Istimulated motility of neuroblastoma cells at a low dose.

The present invention shows that NDGA effectively suppressesneuroblastoma growth in vitro and in vivo, and inhibits the motility andpromotes the apoptosis of neuroblastoma cells in culture. These effectsare attributable, at least in part, to the prevention of IGF-IRactivation by IGFs, an important event in the regulation ofneuroblastoma growth, survival, and motility. The present invention alsoshows that NDGA administration to animals is well-tolerated andeffective. The NDGA formulation of the invention can be co-administeredwith anti-myc agents and/or radiation, so as to be even more effectivein treatments that affect other aspects of neuroblastoma tumorigenesis.NDGA, in combination with other agents that affect IGF action, such asIGF binding proteins or anti-PI-3K agents, can provide increased tumorkill with decreased general toxicities.

Example 3

Materials and methods

Materials

NDGA and IGF-1 were a gift from Insmed Inc. (Glen Allen, Va.).Antibodies against the IGF-1R (C-20), HER2 (C-18), and phosphospecificantibodies recognizing phosphotyrosine (PY20), and pNeu (Tyr1248), andHRP-conjugated anti-phosphotyrosine antibody (PY20HRP) were all obtainedfrom Santa Cruz Biotechnology (Santa Cruz, Calif.). Alpha IR3, amonoclonal antibody against the IGF-1R, was obtained from CalBiochem(San Diego, Calif.). Phosphospecific antibodies pIGF-IR (Y1131) andpAkt(ser473) were obtained from Cell Signaling (Beverly, Mass.). Allother reagents were from Sigma (St. Louis, Mo.), except as indicatedbelow. Gefitinib (Iressa) was a gift of Mark Moasser, University ofCalifornia San Francisco. Herceptin was purchased from a commercialpharmacy.

Cell Culture

MCF-7 cells stably transfected with the full length HER2 cDNA(MCF-7/HER2-18) or control vector (MCF-7/neo) were generously providedby Dr. Christopher Benz (Buck Institute for Age Research, Novato,Calif.) and were maintained at 37 oC, 5% CO2 in DMEM+10% FCS (DMEM-10)supplemented with 200 μg/ml Geneticine.

Preparation of Cell Lysates

For dose effects of RTK inhibitors (NDGA or gefitinib) on cellularIGF-1R and HER2 signaling, cells were grown in 6-well plates to ˜80%confluency, then serum-starved for 18 hr. RTK inhibitors were dissolvedin DMSO and diluted with culture medium before being added to cells for1.5 hour at 37° C. The final concentration of DMSO during the incubationwas 0.3%. For some studies, cells were also stimulated with 3 nM IGF-Ifor 10 minutes at 37° C. Reactions were terminated by rapidly aspiratingmedium and washing cells three times with ice cold PBS. Cells wereharvested and solubilized in 50 mM HEPES, 150 mM NaCl, 1% Triton X-100,1 mM PMSF, and 2 mM vanadate for 1 hour at 4° C. Protein concentrationswere determined by BCA assay (Pierce, Rockford, Ill.). Enzyme linkedimmunosorbent assays (ELISA) for phosphorylated IGF-1R and HER2 IGF-1Rphosphorylation was determined by ELISA as described previously for theinsulin receptor [Youngren J F, Goldfine I D and Pratley R E: Decreasedmuscle insulin receptor kinase correlates with insulin resistance innormoglycemic Pima Indians. Am J Physiol 273: E276-83, 1997]. Briefly,10 μg lysate protein were added to triplicate wells in a 96-well platecoated with monoclonal antibody to the IGF-IR (IR3; 2 μg/ml), andincubated for 18 hours at 4° C. Plates were washed five times and thenHRP-conjugated anti-phosphotyrosine antibody (0.3 μg/ml), diluted inSolution B (50 mM HEPES, pH 7.6, 150 mM NaCl, 0.05% Tween-20, 1 mM PMSF,2 mM vanadate and 1 mg/ml bacitracin), was added for two hours at 22° C.Plates were washed five times prior to color development with TMBsubstrate, which was terminated with 1.0 M H3PO4. Values for receptorphosphorylation were determined by measuring absorbance at 450 nm.

HER2 phosphorylation was also determined by ELISA as above, using 2 μgof lysate protein per well and 2 μg/ml Herceptin as the capturingantibody.

Western Blot Analysis

Total protein extracts (10 μg), prepared as described above from cellscultured in the presence or absence of IGF-1 and/or RTK inhibitors, weresubjected to SDS-PAGE and subsequently transferred to nitrocellulosemembranes. Membranes were incubated overnight at 4 oC with primaryantibody diluted in Superblock (Pierce) containing 0.1% Tween 20(Bio-Rad). The membranes were washed 3 times with TBS-T, then incubatedwith HRP-conjugated secondary antibody diluted in Superblock/Tween 20for 90 min at room temperature. Membranes were washed again and boundantibodies detected by enhanced chemiluminescence (Pierce). Primaryantibodies against the following proteins were used at the indicateddilutions: IGF-1R used at 0.2 μg/ml, HER2 used at 0.2 μg/ml, p-IGF-1R(Y1131) used at 1:1000, pNeu (Tyr1248) used at 0.2 μg/ml, andpAkt(ser473) used at 1:1000. Secondary HRP-conjugated antibodies weredirected against the appropriate species of origin of the primaryantibody.

Cell Growth Assays

The inhibitory effects of RTK inhibitors (NDGA and gefitinib) on breastcancer cell growth were determined using a CyQuant cell proliferationassay kit (Molecular Probes, Eugene, Oreg.). MCF-7/neo or MCF-7/HER2-18cells were plated in 96 well plates (4×103 cells/well) in 100 μl/well ofDMEM-10 medium. Cells were allowed to adhere overnight and were thentreated with various concentrations of NDGA, gefitinib, or DMSO as avehicle control in 100 μl/well of serum free DMEM (SF-DMEM), making thefinal serum concentration 5%. Media with inhibitors was refreshed on day3 and the cultures were harvested on day 6. The plates were invertedonto paper towels with gentle blotting to remove growth medium withoutdisrupting adherent cells. Each plate was kept at −80° C. until assayedfor cell growth. After thawing the plate at room temperature, 200 μl ofCyQuant GR solution was added to each well and the plates were incubatedin the dark for five minutes. Fluorescence was measured with aSpectraMax Gemini XS fluorescence microplate reader (Molecular Devices)with 480-nm excitation and 520-nm emission.

The growth inhibitory effects of tamoxifen, in the presence or absenceof NDGA, were assessed in MCF-7/HER2-18 cells using a CyQuant assay asdescribed above with a few modifications to the protocol. Cells wereestrogen-starved for three days in DMEM containing 10%charcoal-dextrin-stripped FCS (CDSS) prior to their plating in 96 wellplates. Cells were plated in 100 μl of the same media, allowed to adhereovernight, and then were switched to DMEM+10% CDSS supplemented with 100pM estrogen. Tamoxifen (100 nM final concentration) and/or NDGA (10-20μM final concentration) was added in 100 μl of SF-DMEM to yield a finalconcentration of 5% CDSS in DMEM. The media and inhibitors wererefreshed on day 3, the cultures were harvested on day 6, and a CyQuantassay was performed as described above.

Results HER2 Receptor, But Not the IGF-1R, is Overexpressed in TamoxifenResistant MCF-7/HER2-18 Cells.

Various studies have demonstrated reduced IGF-1R expression inantiestrogen-resistant cell lines [Brockdorff B L, Heiberg I andLykkesfeldt A E: Resistance to different antiestrogens is caused bydifferent multi-factorial changes and is associated with reducedexpression of IGF receptor Ialpha. Endocr Relat Cancer 10: 579-90, 2003;Frogne T, Jepsen J S, Larsen S S, Fog C K, Brockdorff B L andLykkesfeldt A E: Antiestrogen-resistant human breast cancer cellsrequire activated protein kinase B/Akt for growth. Endocr Relat Cancer12: 599-614, 2005; McCotter D, van den Berg H W, Boylan M and McKibbenB: Changes in insulin-like growth factor-I receptor expression andbinding protein secretion associated with tamoxifen resistance andestrogen independence in human breast cancer cells in vitro. Cancer Lett99: 239-45, 1996; van den Berg H W, Claffie D, Boylan M, McKillen J,Lynch M and McKibben B: Expression of receptors for epidermal growthfactor and insulin-like growth factor I by ZR-75-1 human breast cancercell variants is inversely related: the effect of steroid hormones oninsulin like growth factor I receptor expression. Br J Cancer 73:477-81, 1996]. We examined the levels of the IGF-1R and HER2 proteins inparental MCF-7/neo and MCF-7/HER2-18 cells using Western blot analyses.Compared to the MCF-7/neo cells, the level of IGF-1R was decreased inMCF-7/HER2-18 cells (FIG. 22), consistent with the reports describedabove.

In contrast, the HER2 protein was abundant in the MCF-7/HER2-18 cellsbut undetectable in MCF-7/neo cells (FIG. 22). This observation is inagreement with the original report that MCF-7/HER2-18 cells overexpressthe HER2 protein compared to parental MCF-7 cells [Benz C C, Scott G K,Sarup J C, Johnson R M, Tripathy D, Coronado E, Shepard H M and OsborneC K: Estrogen-dependent, tamoxifen-resistant tumorigenic growth of MCF-7cells transfected with HER2/neu. Breast Cancer Res Treat 24: 85-95,1993].

NDGA, But Not Gefitinib, Equally Inhibits the Growth of Both Parentaland Tamoxifen Resistant MCF-7/HER2-18 Cells

Gefitinib, an EGFR inhibitor, has been shown to inhibit the growth ofHER2-overexpressin breast cancer cell lines, including MCF-7/HER2-18[Anderson N G, Ahmad T, Chan K, Dobson R and Bundred N J: ZD1839(Iressa), a novel epidermal growth factor receptor (EGFR) tyrosinekinase inhibitor, potently inhibits the growth of EGFR-positive cancercell lines with or without erbB2 overexpression. Int J Cancer 94:774-82, 2001; Moasser M M, Basso A, Averbuch S D and Rosen N: Thetyrosine kinase inhibitor ZD1839 (“Iressa”) inhibits HER2-drivensignaling and suppresses the growth of HER2-overexpressina tumor cells.Cancer Res 61: 7184-8, 2001; Moulder S L, Yakes F M, Muthuswamy S K,Bianco R, Simpson J F and Arteaga C L: Epidermal growth factor receptor(HER1) tyrosine kinase inhibitor ZD1839 (Iressa) inhibits HER2/neu(erbB2)-overexpressing breast cancer cells in vitro and in vivo. CancerRes 61: 8887-95, 2001]. NDGA inhibits both HER2 and IGF-1R activitiesand has been shown to inhibit the growth of HER2-negative (MCF-7) aswell as HER2-positive (SKBr3) breast cancer cells [Youngren J F, GableK, Penaranda C, Maddux B A, Zavodovskaya M, Lobo M, Campbell M, Kerner Jand Goldfine I D: Nordihydroguaiaretic acid (NDGA) inhibits the IGF-1and cerbB2/HER2/neu receptors and suppresses growth in breast cancercells. Breast Cancer Res Treat 94: 37-46, 2005]. We compared the effectsof both tyrosine kinase inhibitors on the growth of parental MCF-7/neoand MCF-7/HER2-18 cells. Gefitinib (FIG. 23A) was more effective atinhibiting the growth of HER2-overexpressing MCF-7/HER2-18 cells whencompared to MCF-7/neo cells. In contrast, NDGA was equally effective inboth cells lines, with a one half maximal effect occurring at 12.5 μM(FIG. 23B).

NDGA Inhibits Both the IGF-1R and HER2 Receptor and Downstream Signalingin Tamoxifen Resistant MCF-7/HER2-18 Cells

The tamoxifen resistant MCF-7/HER2-18 cell line expresses both IGF-1Rand HER2, two receptor tyrosine kinases (RTK) that play a role in breastcancer. We have previously shown that NDGA inhibits the kinaseactivities of IGF-1R in MCF-7 cells and HER2 in SKBR-3 cells [Youngren JF, Gable K, Penaranda C, Maddux B A, Zavodovskaya M, Lobo M, Campbell M,Kerner J and Goldfine I D: Nordihydroguaiaretic acid (NDGA) inhibits theIGF-1 and cerbB2/HER2/neu receptors and suppresses growth in breastcancer cells. Breast Cancer Res Treat 94: 37-46, 2005]. We next comparedthe effects of NDGA with gefitinib on the activities of these receptorkinases in MCF-7/HER2-18 cells.

Employing a sensitive ELISA [Youngren J F, Goldfine I D and Pratley R E:Decreased muscle insulin receptor kinase correlates with insulinresistance in normoglycemic Pima Indians. Am J Physiol 273: E276-83,1997] we observed that gefitinib had only a weak inhibitory effect onIGF-1 stimulated phosphorylation of the IGF-1R (FIG. 24A). In contrast,gefitinib significantly inhibited HER2 phosphorylation with a halfmaximal effect occurring at less than 5 μM (FIG. 24A). Whereas gefitinibsuppressed HER2 but not IGF-1R activity, NDGA strongly inhibited bothkinases as measured by phosphotyrosine specific ELISAs (FIG. 24B) and byWestern blot (FIG. 24B, inset).

The serine kinase AKT/PKB is activated by receptor tyrosine kinases,including IGF-1R and HER2, and mediates cell growth [Ahmad S, Singh Nand Glazer R I: Role of AKT1 in 17 beta-estradiol- and insulin-Ekegrowth factor I (IGF-I)-dependent proliferation and prevention ofapoptosis in MCF-7 breast carcinoma cells. Biochem Pharmacol 58: 425-30,1999; Martin M B, Franke T F, Stoica G E, Chambon P, Katzenellenbogen BS, Stoica B A, McLemore M S, Olivo S E and Stoica A: A role for Akt inmediating the estrogenic functions of epidermal growth factor andinsulin-like growth factor I. Endocrinology 141: 4503-11, 2000;Mitsiades C S, Mitsiades N and Koutsilieris M: The Akt pathway:molecular targets for anticancer drug development. Curr Cancer DrugTargets 4: 235-56, 2004: Stoica G E, Franke T F, Wellstein A, CzubaykoF, List H J, Reiter R, Morgan E, Martin M B and Stoica A: Estradiolrapidly activates Akt via the ErbB2 signaling pathway. Mol Endocrinol17: 818-30, 2003]. We measured the effects of NDGA on the phosphorylatedstate of this protein in MCF-7/HER2-18 cells. In the absence of IGF-1,AKT/PKB was phosphorylated and the level of phospho-AKT/PKB wasdecreased by treatment with NDGA (FIG. 25). Addition of 3 nM IGF-1increased the amount of phospho-AKT/PKB and NDGA also inhibited thisIGF-1 stimulated phosphorylation of AKT/PKB. NDGA did not alter thecontent of the AKT/PKB protein in MCF-7/HER2-18 cells (data not shown).

NDGA Attenuates Tamoxifen Resistance in HER2 Overexpressing MCF-7 Cells

Several reports have demonstrated cross-talk between IGF-1R and HER2signaling pathways [Gee J M, Robertson J F, Gutteridge E, Ellis I O,Pinder S E, Rubini M and Nicholson R I: Epidermal growth factorreceptor/HER2/insulin-like growth factor receptor signalling andoestrogen receptor activity in clinical breast cancer. Endocr RelatCancer 12 Suppl 1: S99-S111, 2005; Lu Y, Zi X, Zhao Y and Pollak M:Overexpression of ErbB2 receptor inhibits IGF-I induced Shc-MAPKsignaling pathway in breast cancer cells. Biochem Biophys Res Commun313: 709-15, 2004; Nahta R, Yuan L X, Zhang B, Kobayashi R and Esteva FJ: Insulin-like growth factor-I receptor/human epidermal growth factorreceptor 2 heterodimerization contributes to trastuzumab resistance ofbreast cancer cells. Cancer Res 65: 11118-28, 2005] as well as betweenER signaling and these RTKs [Lee A V, Weng C N, Jackson J G and Yee D:Activation of estrogen receptor-mediated gene transcription by IGF-I inhuman breast cancer cells. J Endocrinol 152: 39-47, 1997: Martin M B andStoica A: Insulin-like growth factor-I and estrogen interactions inbreast cancer. J Nutr 132: 3799S-3801S, 2002; Yee D and Lee A V:Crosstalk between the insulin-like growth factors and estrogens inbreast cancer. J Mammary Gland Biol Neoplasia 5: 107-15, 2000]. Giventhis cross-talk, we next examined whether NDGA (which inhibits both HER2and IGF-1R activities) could overcome tamoxifen resistance inMCF-7/HER2-18 cells.

Tamoxifen at 100 nM inhibited the growth of MCF-7/neo cells by over 50%(FIG. 26A). In contrast, tamoxifen at 100 nM had less of an effect onMCF-7/HER2-18 cells (24% growth inhibition) (FIG. 26B), consistent withother reports demonstrating tamoxifen resistance of MCF-7/HER2-18 cells[Benz C C, Scott G K, Sarup J C, Johnson R M, Tripathy D, Coronado E,Shepard H M and Osborne C K: Estrogen-dependent, tamoxifen-resistanttumorigenic growth of MCF-7 cells transfected with HER2/neu. BreastCancer Res Treat 24: 85-95, 1993].

Both NDGA and tamoxifen had antiproliferative effects on the tamoxifensensitive MCF-7/neo cells (FIG. 26A). NDGA at 10 and 15 μM inhibitedgrowth by 23% and 55%, respectively. However, when combined, NDGAtreatment did not enhance the growth inhibitory effects of tamoxifen inthese cells (FIG. 26C). In contrast to MCF-7/neo cells, NDGA treatmentsignificantly enhanced the antiproliferative effects of tamoxifen inthese anti-estrogen resistant cells (FIG. 26D). NDGA alone, at 10 and 15μM, inhibited the growth of MCF-7/HER2-18 cells by 9 and 38%,respectively. While tamoxifen alone induced a 24% reduction in growth,the combination of tamoxifen with 10 or 15 μM NDGA resulted in 40 and60% growth inhibition, respectively, indicating additive effects of NDGAand tamoxifen.

Discussion

Interference with growth factor signals that drive cell proliferationand survival is an attractive strategy for cancer treatment. Initialexplorations have concentrated on types of cancers in which growthfactor signaling is elevated and plays a dominant role in driving cellproliferation. A well known example is breast cancer with overexpressionof the HER2 receptor which responds to interventions that block HER2action, such as gefitinib [Benz C C, Scott G K, Sarup J C, Johnson R M,Tripathy D, Coronado E, Shepard H M and Osborne C K: Estrogen-dependent,tamoxifen-resistant tumorigenic growth of MCF-7 cells transfected withHER2/neu. Breast Cancer Res Treat 24: 85-95, 1993; Agrawal A, GutteridgeE, Gee J M, Nicholson R I and Robertson J F: Overview of tyrosine kinaseinhibitors in clinical breast cancer. Endocr Relat Cancer 12 Suppl 1:S135-44, 2005; Arteaga C L, Moulder S L and Yakes F M: HER (erbB)tyrosine kinase inhibitors in the treatment of breast cancer. SeminOncol 29: 4-10, 2002; Arteaga C L and Truica C I: Challenges in thedevelopment of anti-epidermal growth factor receptor therapies in breastcancer. Semin Oncol 31: 3-8, 2004; Herbst R S and Kies M S: Gefitinib:current and future status in cancer therapy. Clin Adv Hematol Oncol 1:466-72, 2003; Johnston S R: Clinical trials of intracellular signaltransductions inhibitors for breast cancer—a strategy to overcomeendocrine resistance. Endocr Relat Cancer 12 Suppl 1: S145-57, 2005:Kaklamani V and O'Regan R M: New targeted therapies in breast cancer.Semin Oncol 31: 20-5, 2004; Konecny G E, Wilson C A and Slamon D J: Isthere a role for epidermal growth factor receptor inhibitors in breastcancer prevention? J Natl Cancer Inst 95: 1813-5, 2003; Penne K, BohlinC, Schneider S and Allen D: Gefitinib (Iressa, ZD1839) and tyrosinekinase inhibitors: the wave of the future in cancer therapy. Cancer Nurs28: 481-6, 2005; Von Pawel J: Gefitinib (Iressa, ZD1839): a noveltargeted approach for the treatment of solid tumors. Bull Cancer 91:E70-6, 2004; Wakeling A E: Inhibitors of growth factor signalling.Endocr Relat Cancer 12 Suppl 1: S183-7, 2005] or Herceptin [Baselga J,Carbonell X, Castaneda-Soto N J, Clemens M, Green M, Harvey V, MoralesS, Barton C and Ghahramani P: Phase II study of efficacy, safety, andpharmacokinetics of trastuzumab monotherapy administered on a 3-weeklyschedule. J Clin Oncol 23: 2162-71, 2005; Marty M, Cognetti F,Maraninchi D, Snyder R, Mauriac L, Tubiana-Hulin M, Chan S, Grimes D,Anton A, Lluch A, Kennedy J, O'Byrne K, Conte P, Green M, Ward C, MayneK and Extra J M: Randomized phase II trial of the efficacy and safety oftrastuzumab combined with docetaxel in patients with human epidermalgrowth factor receptor 2-positive metastatic breast cancer administeredas first-line treatment: the M77001 study group. J Clin Oncol 23:4265-74, 2005; Slamon D J, Leyland-Jones B, Shak S, Fuchs H, Paton V,Bajamonde A, Fleming T, Eiermann W, Wolter J, Pegram M, Baselga J andNorton L: Use of chemotherapy plus a monoclonal antibody against HER2for metastatic breast cancer that overexpresses HER2. N Engl J Med 344:783-92, 2001; Vogel C L, Cobleigh M A, Tripathy D, Gutheil J C, Harris LN, Fehrenbacher L, Slamon D J, Murphy M, Novotny W F, Burchmore M, ShakS, Stewart S J and Press M: Efficacy and safety of trastuzumab as asingle agent in first-line treatment of HER2-overexpressing metastaticbreast cancer. J Clin Oncol 20: 719-26, 2002]. Herein we have examinedthe action of NDGA, an agent that blocks signaling through HER2receptors, as well as IGF-1 receptors [Youngren J F, Gable K, PenarandaC, Maddux B A, Zavodovskaya M, Lobo M, Campbell M, Kerner J and GoldfineI D: Nordihydroguaiaretic acid (NDGA) inhibits the IGF-1 andcerbB2/HER2/neu receptors and suppresses growth in breast cancer cells.Breast Cancer Res Treat 94: 37-46, 2005], and compared it with that ofgefitinib which is directed exclusively at EGFR/HER2 receptors. Both ofthese drugs work at the level of preventing receptorauto-phosphorylation, a factor that was monitored in our studies.

We studied the actions of these drugs on MCF-7/neo cells, an estrogenreceptor positive human breast cancer cell line that is sensitive to theantiestrogen tamoxifen, and MCF-7/HER2-18 cells that overexpress theHER2 receptor and demonstrate a reduced sensitivity to tamoxifen.MCF-7/neo cells express IGF-1 receptors but not HER2, whereas theMCF-7/HER2-18 cell line expresses both IGF-1R and HER2.

Our studies show that gefitinib, as expected from its mode of action,inhibits the growth of MCF-7/HER2-18 cells, but has less potency onMCF-7/neo cells. These observations are consistent with the notion thatinhibition of HER2 receptors is likely to be most effective on cellswhose proliferation is driven by enhanced activity of this receptorpathway. In contrast to the selective efficacy of gefitinib, we foundthat NDGA is equally effective in inhibiting cell proliferation of bothMCF-7/neo cells and MCF-7/HER2-18 cells. Inhibition of MCF-7/HER2-18cells by NDGA is accompanied by an increase in apoptosis (data notshown) as well as a decrease in downstream signaling (Aktphosphorylation). The kinetics of inhibition of cell proliferation ofMCF-7/HER2-18 cells match those of NDGA inhibition of IGF-1 receptorphosphorylation with half-maximal effects in the range of 10-20 μM.Inhibition of HER2 receptor phosphorylation was also evident, withhalf-maximal effects in the range of 30 μM. Breast cancers are eitherestrogen-dependent or -independent. A subset of breast cancers, despitethe presence of estrogen receptors, do not respond to endocrine therapyand it has been reported that HER2 expression is associated with areduced response rate to hormone therapy of metastatic breast cancer[Wright C, Angus B, Nicholson S, Sainsbury J R, Cairns J, Gullick W J,Kelly P, Harris A L and Home C H: Expression of c-erbB-2 oncoprotein: aprognostic indicator in human breast cancer. Cancer Res 49: 2087-90,1989]. Transfection of ER-positive cells with a HER2 cDNA, resulting inoverexpression of this RTK, also results in resistance to tamoxifentreatment [Benz C C, Scott G K, Sarup J C, Johnson R M, Tripathy D,Coronado E, Shepard H M and Osborne C K: Estrogen-dependent,tamoxifen-resistant tumorigenic growth of MCF-7 cells transfected withHER2/neu. Breast Cancer Res Treat 24: 85-95, 1993]. The mechanism ofthis resistance includes signaling from the HER2 receptor to ER alphawhich results in phosphorylation and enhanced action of the hormoneindependent transcriptional activation function one (AF1) of thereceptor. Additionally HER2 signaling leads to phosphorylation andenhanced action of the major coactivator for ERalpha driven geneexpression, the amplified in breast cancer 1 (AIB 1) coactivator [ShouJ, Massarweh S, Osborne C K, Wakeling A E, Ali S, Weiss H and Schiff R:Mechanisms of tamoxifen resistance: increased estrogen receptor-HER2/neucross-talk in ER/HER2-positive breast cancer. J Natl Cancer Inst 96:926-35, 2004]. In addition to these effects downstream of HER2, a feedforward loop in which the liganded ERalpha stimulates the HER2 pathwayis activated. The result is that tamoxifen mimics estrogen and drivesproliferation.

In view of the disruption of tamoxifen action in HER2-overexpressingcells, studies have examined the effects of blocking HER2 signaling intamoxifen resistant breast cancer cells. It has been shown thatgefitinib can overcome tamoxifen resistance, or prevent its development,both in vitro and in a mouse xenograft model [Shou J, Massarweh S,Osborne C K, Wakeling A E, All S, Weiss H and Schiff R: Mechanisms oftamoxifen resistance: increased estrogen receptor-HER2/neu cross-talk inER/HER2-positive breast cancer. J Natl Cancer Inst 96: 926-35, 2004; GeeJ M, Harper M E, Hutcheson I R, Madden T A, Barrow D, Knowlden J M,McClelland R A, Jordan N, Wakeling A E and Nicholson R I: Theantiepidermal growth factor receptor agent gefitinib (ZD1839/Iressa)improves antihormone response and prevents development of resistance inbreast cancer in vitro. Endocrinology 144: 5105-17, 2003; Kurokawa H andArteaga C L: Inhibition of erbB receptor (HER) tyrosine kinases as astrategy to abrogate antiestrogen resistance in human breast cancer.Clin Cancer Res 7: 4436s-4442s; discussion 4411s-4412s, 2001]. However,continuous treatment eventually leads to acquired resistance togefitinib which is associated with increased signaling via the IGF-1R[Jones H E, Goddard L, Gee J M, Hiscox S, Rubini M, Barrow D, Knowlden JM, Williams S, Wakeling A E and Nicholson R I: Insulin-like growthfactor-I receptor signalling and acquired resistance to gefitinib(ZD1839; Iressa) in human breast and prostate cancer cells. Endocr RelatCancer 11: 793-814, 2004].

In addition to interactions between ER and HER2 signaling pathways,cross-talk between IGF-1R and HER2 in breast cancer cells has beenreported [Gee J M, Robertson J F, Gutteridge E, Ellis I O, Pinder S E,Rubini M and Nicholson R I: Epidermal growth factorreceptor/HER2/insulin-like growth factor receptor signalling andoestrogen receptor activity in clinical breast cancer. Endocr RelatCancer 12 Suppl 1: S99-S111, 2005; Lu Y, Zi X, Zhao Y and Pollak M:Overexpression of ErbB2 receptor inhibits IGF-I induced Shc-MAPKsignaling pathway in breast cancer cells. Biochem Biophys Res Commun313: 709-15, 2004; Nahta R, Yuan L X, Zhang B, Kobayashi R and Esteva FJ: Insulin-like growth factor-I receptor/human epidermal growth factorreceptor 2 heterodimerization contributes to trastuzumab resistance ofbreast cancer cells. Cancer Res 65: 11118-28, 2005]. We also haveobserved of cross-talk between IGF-1R and HER2 in MCF-7/HER2-18 cells(unpublished data). Given that IGF-1R signaling plays a role in thedevelopment of gefitnib resistance in tamoxifen resistant cells, andthat there is cross-talk between ER, IGF-1R, and HER2 signalingpathways, we examined the interaction of NDGA (which inhibits bothIGF-1R and HER2) with tamoxifen on the growth of tamoxifen resistantMCF-7/HER2-18 cells. NDGA alone inhibited the proliferation of bothtamoxifen sensitive MCF-7/neo and tamoxifen resistant MCF-7/HER2-18cells. Notably, NDGA combined with tamoxifen demonstrated additivegrowth inhibitory effects on MCF-7/HER2-18 cells. The development ofacquired resistance to anti-hormonal therapies such as tamoxifen is amajor therapeutic problem in breast cancer. These results suggest thatNDGA might be clinically useful, in conjunction with anti-hormonalagents, in the treatment of hormone-resistant breast cancer, or possiblyin preventing the development of acquired resistance to these agents.

In summary, we demonstrated that NDGA inhibits the kinase activities ofthe IGF-1 receptor and the HER2 receptor and blocks cellularproliferation of both MCF-7/neo and MCF-7/HER2-18 cells. NDGA alsoattenuated tamoxifen resistance in the HER2-overexpressing MCF-7/HER2-18cell line. These data raise the possibility that NDGA, and similaragents that target multiple growth factor pathways, will have a broadspectrum of action on breast cancers with a variety of perturbations intheir signaling pathways.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1.-26. (canceled)
 27. A method of treating a human pancreatic cancerpatient comprising: orally administering to the patient atherapeutically effective amount of a formulation comprising: (1) apharmaceutically acceptable carrier; (2) nordihydroguaiaretic acid orpharmaceutically acceptable salts thereof; and (3) doxorubicin, whereinthe NDGA and doxorubicin act synergistically to inhibit growth of thepancreatic cancer cells, and wherein doxorubicin and NDGA areadministered in amounts below a therapeutically effective amount ofeither doxorubicin or NDGA when doxorubicin or NDGA are administeredalone.
 28. The method of claim 27, wherein the amount of doxorubicinadministered is less than 60 mg/m² once in 21 days, or less than 30mg/m² daily for 3 days every four weeks.
 29. The method of claim 27,wherein the amount of NDGA administered is less than 100 mg/kg orallythree times a week, or 37.5 mg/kg intraperitoneally three times a week.30. The method of claim 27, wherein side effects are decreased comparedto giving either doxorubicin or NDGA at higher dosages.
 31. A method oftreating a human pancreatic cancer patient, wherein pancreatic cancercells in the cancer patient do not express Her2/neu receptor,comprising: orally administering to the patient a therapeuticallyeffective amount of a formulation comprising: (1) a pharmaceuticallyacceptable carrier; (2) nordihydroguiaretic acid (NDGA) orpharmaceutically acceptable salts thereof; (3) and doxorubicin, whereinthe NDGA and doxorubicin act synergistically to inhibit growth of thepancreatic cancer cells, and wherein doxorubicin and NDGA areadministered in amounts below a therapeutically effective amount ofeither doxorubicin or NDGA when doxorubicin or NDGA are administeredalone.
 32. The method of claim 31, wherein the amount of doxorubicinadministered is less than 60 mg/m² once in 21 days, or less than 30mg/m² daily for 3 days every four weeks.
 33. The method of claim 31,wherein the amount of NDGA administered is less than 100 mg/kg orallythree times a week, or 37.5 mg/kg intraperitoneally three times a week.34. The method of claim 31, wherein side effects are decreased comparedto giving either doxorubicin or NDGA at higher dosages.
 35. A method oftreating a pancreatic cancer patient, wherein pancreatic cancer cells inthe cancer patient express Her2/neu receptor, comprising the steps of:administering to the patient a therapeutically effective amount of aformulation comprising: (1) a pharmaceutically acceptable carrier; (2)nordihydroguaiaretic acid (NDGA) or pharmaceutically acceptable saltsthereof; and (3) doxorubicin, wherein the NDGA and doxorubicin actsynergistically to inhibit growth of the pancreatic cancer cells, andwherein the amount of doxorubicin administered is less than 60 mg/m²once in 21 days, or less than 30 mg/m² daily for 3 days every four weeksand the amount of NDGA is less than 100 mg/kg orally three times a week,or 37.5 mg/kg intraperitoneally three times a week.
 36. A method oftreating a pancreatic cancer patient, wherein pancreatic cancer cells inthe cancer patient do not express Her2/neu receptor, comprising thesteps of: administering to the patient a therapeutically effectiveamount of a formulation comprising: (1) a pharmaceutically acceptablecarrier; (2) nordihydroguaiaretic acid (NDGA) or pharmaceuticallyacceptable salts thereof; and (3) doxorubicin, wherein the NDGA anddoxorubicin act synergistically to inhibit growth of the pancreaticcancer cells, and wherein the amount of doxorubicin administered is lessthan 60 mg/m² once in 21 days, or less than 30 mg/m² daily for 3 daysevery four weeks and the amount of NDGA is less than 100 mg/kg orallythree times a week, or 37.5 mg/kg intraperitoneally three times a week.